Photoeradication of microorganisms with pulsed purple or blue light

ABSTRACT

The present invention is directed to a system and method for photoeradication of microorganisms from a target. The method includes the step of obtaining test data for a plurality of experiments each of which comprises irradiating test microorganisms with a plurality of light pulses having a wavelength that ranges from 380 nm to 500 nm. The light pulses have a plurality of pulse parameters (peak irradiance, pulse duration, and off time between adjacent light pulses) and are provided at a radiant exposure that ranges from 0.5 J/cm 2  to 60 J/cm 2  during each of a plurality of irradiation sessions. The test data comprises a survival rate for the test microorganisms after irradiation with the light pulses. The method also includes the step of analyzing the test data to identify the pulse parameters for the light pulses and the radiant exposure for each of the irradiation sessions that result in a desired survival rate for the test microorganisms. The method further includes the step of irradiating the microorganisms of the target with light pulses having the identified pulse parameters at the identified radiant exposure for each of the irradiation sessions so as to photoeradicate all or a portion of the microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims priority toU.S. patent application Ser. No. 16/205,182, filed on Nov. 29, 2018,which is a continuation-in-part of and claims priority to U.S. patentapplication Ser. No. 15/955,773, filed on Apr. 18, 2018, which is acontinuation of and claims priority to PCT Patent Application Serial No.PCT/US2017/034396, filed on May 25, 2017, which is based on and claimspriority to U.S. Provisional Application Ser. No. 62/341,691, filed onMay 26, 2016, each of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

STATEMENT REGARDING JOINT RESEARCH AGREEMENT

Not applicable.

BACKGROUND OF THE INVENTION

Various devices for delivering light to a region of skin for therapeuticor cosmetic purposes are known in the art. The use of phototherapy, inparticular blue light, as an armamentarium for antimicrobial activityhas been of great interest, particularly since Enwemeka et al., firstreported in 2007 that 405 nm and 470 nm light inactivate themethicillin-resistant Staphylococcus aureus (MRSA) bacteria. Currentresearch indicates that bacteria kill rate is tied to the intensity andtotal amount of irradiation energy; that is, the higher the intensityused and the higher the total energy of irradiation, the better thebacteria kill rate. For example, conventional light emitting diodes(LEDs) operating in a continuous wave (CW) mode of irradiation have beenused to deliver light at high irradiances and radiant exposures toincrease bacteria kill rates. However, there is a risk that these highirradiances and radiant exposures may damage other tissues in the regionunder treatment through thermal or photochemical effects or may providea significant optical hazard to the subject undergoing treatment.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a system and method forphotoeradication of microorganisms from a target using pulsed purple orblue light. In some embodiments, the target includes an endogenous orexogenous photoactive molecule in which (a) the light pulses have a peakirradiance and a pulse duration sufficient to optically excite thephotoactive molecule and (b) the light pulses are separated by an offtime sufficient to allow the photoactive molecule to return to a groundstate creating an oxidation reaction that produces free radicals whichdestroy a cellular structure of all or a portion of the microorganisms.In other embodiments, the target is irradiated with pulsed purple orblue light so as to destroy the RNA or DNA genomes of all or a portionof the microorganisms.

In a first embodiment, the target comprises a region of skin, tissue, awound, or cells of a human or animal body that are infected by varioustypes of microorganisms, such as bacteria, viruses or fungi. Theinfected skin, tissue, wound or cells are treated by irradiating thetarget with pulsed purple or blue light. Preferably, the pulsed purpleor blue light is provided at specified pulse parameters, dosages andtime intervals so as to inactivate all or a portion of themicroorganisms at low irradiances and radiant exposures compared tothose of a continuous wave (CW) mode of irradiation. As a result, it isbelieved that the microorganisms can be inactivated without damage toother tissues or cells in the region under treatment. In some cases, thetarget is a host cell subject to viral invasion that is destroyedwithout disrupting non-invaded cells.

In a second embodiment, the target comprises an environment contaminatedwith various types of microorganisms, such as methicillin-resistantStaphylococcus aureus (MRSA) or viruses such as SARS-CoV, 2019-nCoV,human coronavirus 229E, bat coronavirus HKU9-1, H1N1, norovirus (NoV)and feline calicivirus (which are part of the Caliciviridae family),influenza virus type A, HCoV-OC43, and HCoV-NL63. The contaminatedenvironment may comprise, for example, a locker room, a public orprivate restroom, an airplane, a school, a beach, a playground, aplaying field, a hospital or a clinical environment. The contaminatedenvironment is irradiated with pulsed purple or blue light as discussedabove so as to photoeradicate the microorganisms and thereby sanitizethe area.

In a third embodiment, the target comprises food under storage ortransport conditions that is contaminated with microorganisms, such asSalmonella spp., E. Coli, Listeria spp, norovirus (NoV) (which is partof the Caliciviridae family), or hepatitis A. The food may be containedwithin, for example, a refrigeration system, a food display system, afood storage area, or a food processing system. The food is irradiatedwith pulsed purple or blue light as discussed above so as tophotoeradicate the microorganisms and thereby enhance shelf-life andreduce the potential for significant infection transmission to human andanimal populations.

BRIEF DESCRIPTION OF THE DRAWINGS

Various exemplary embodiments of the present invention are described indetail below with reference to the attached drawing figures, asdescribed below.

FIG. 1 is a graph depicting P. acnes survival when irradiated with 450nm light at radiant exposures of 20 and 60 J/cm²; irradiance of 4.5mW/cm²; continuous wave (CW) mode of irradiation; and single irradiationat 0 hours.

FIG. 2 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 20 J/cm²; irradiance of 4.5 mW/cm²;continuous wave (CW) mode of irradiation; and multiple irradiation at 0,24 and 48 hours.

FIG. 3 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 20 J/cm²; irradiance of 4.5 mW/cm²;continuous wave (CW) mode of irradiation; and multiple irradiation at 0,4, 24 and 48 hours.

FIG. 4 is a graph depicting P. acnes survival when irradiated with 450nm light at radiant exposures of 30, 40, 45 and 60 J/cm²; irradiance of4.5 mW/cm²; continuous wave (CW) mode of irradiation; and multipleirradiation at 0 and 4 hours.

FIG. 5 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 60 J/cm²; average irradiance of 5mW/cm²; continuous wave (CW) mode of irradiation and pulsed mode ofirradiation (33% duty factor); and multiple irradiation at 0 and 4hours.

FIG. 6 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 20 J/cm²; average irradiance of 5mW/cm²; continuous wave (CW) mode of irradiation and pulsed mode ofirradiation (33% and 20% duty factors); and multiple irradiation at 0and 4 hours.

FIG. 7 is a graph depicting P. acnes survival when irradiated with 450nm light at radiant exposures of 5, 10 and 20 J/cm²; average irradianceof 2 mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 4, 24 and 48 hours.

FIG. 8 is a graph depicting P. acnes survival when irradiated with 450nm light at radiant exposures of 5, 10 and 20 J/cm²; average irradianceof 3.5 mW/cm²; pulsed mode of irradiation (33% duty factor); andmultiple irradiation at 0, 4, 24 and 48 hours.

FIG. 9 is a graph depicting P. acnes survival when irradiated with 450nm light at radiant exposures of 5, 10 and 20 J/cm²; irradiance of 5mW/cm²; continuous wave (CW) mode of irradiation; and multipleirradiation at 0, 4, 24 and 48 hours.

FIG. 10 is a graph depicting P. acnes survival when irradiated with 450nm light at radiant exposures of 5, 10 and 20 J/cm²; average irradianceof 3 mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 4, 24 and 48 hours.

FIG. 11 is a graph depicting P. acnes survival when irradiated with 450nm light at radiant exposures of 5, 10 and 20 J/cm²; average irradianceof 3 mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 6 and 24 hours.

FIG. 12 is a graph depicting P. acnes survival when irradiated with 450nm light at radiant exposures of 5, 10 and 20 J/cm²; average irradianceof 3 mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3 and 6 hours.

FIG. 13 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 5 J/cm²; average irradiance of 3mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours.

FIG. 14 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 5 J/cm²; average irradiance of 3mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78 hours.

FIG. 15 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 5 J/cm²; average irradiance of 2mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78 hours.

FIG. 16 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 5 J/cm²; average irradiance of 2mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours.

FIG. 17 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 3.6 J/cm²; average irradiance of 2mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 24, 27, 48, 51, 72 and 75 hours.

FIG. 18 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 3.6 J/cm²; average irradiance of 2mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 24, 27, 48, 51, 72, 75, 96 and 99 hours.

FIG. 19 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 3.6 J/cm²; average irradiance of 2mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78 hours.

FIG. 20 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 5 J/cm²; average irradiance of 2mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours.

FIG. 21 is a graph depicting P. acnes survival when irradiated with 450nm light at a radiant exposure of 3.6 J/cm²; average irradiance of 2mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours.

FIG. 22 illustrates a plurality of light emitting diodes printed on aflexible film (referred to herein as a printed LED flexible lamp or alighted substrate) that is suitable for use in accordance with thepresent invention;

FIG. 23 is a schematic illustration depicting an experimental set-upused in bacteria irradiation with a 458 nm laser light.

FIG. 24A is a graph depicting porphyrin emission extracted from P. acnesexcited with 458 nm light.

FIG. 24B is a graph depicting porphyrin fluorescence emission extractedfrom P. acnes excited with 450 nm light from the printed LED flexiblelamp shown in FIG. 22 at an average irradiance of 3 mW/cm²; pulsed modeof irradiation (33% duty factor); and multiple irradiation at 0, 10, 20and 30 minutes.

FIG. 24C is a graph depicting porphyrin emission extracted from a sampleof Protoporphyrin IX excited with 450 nm light from the printed LEDflexible lamp shown in FIG. 22.

FIG. 25 illustrates the general structure of a light source in the formof a very thin layered structure that is suitable for delivering pulsedlight in accordance with the present invention.

FIGS. 26A and 26B illustrate various exemplary shapes of light sourcesthat are suitable for use in the light source of FIG. 25.

FIG. 27 illustrates an exemplary OLED structure for use in the lightsource of FIG. 25.

FIG. 28 illustrates an exemplary printable LED structure for use in thelight source of FIG. 25.

FIG. 29 illustrates a light source with a bottom light emittingconfiguration that is suitable for delivering pulsed light in accordancewith the present invention.

FIG. 30 illustrates a light source with a top light emittingconfiguration that is suitable for delivering pulsed light in accordancewith the present invention.

FIG. 31 is a block diagram of an electronic circuit for controlling alight source in accordance with the present invention, wherein theelectronic circuit includes a drive circuit and microcontroller that ispreprogrammed to provide a fixed dose of light.

FIG. 32 is a block diagram of an electronic circuit for controlling alight source in accordance with the present invention, wherein theelectronic circuit includes sensors that operate in a closed loop toprovide feedback to a microcontroller so as to dynamically control thelight source.

FIGS. 33 and 34 are graphs depicting GBS survival for differentcombinations of 0.002, 0.02 or 0.05 mg/mL PPIX supplementation and/orirradiation with 450 nm light at a radiant exposure of 7.56 J/cm²;average irradiance of 3 mW/cm²; pulsed mode of irradiation (33% dutyfactor); and multiple irradiation at 0, 72 and 144 hours.

FIG. 35 is a graph depicting GBS survival for different combinations of0.002 or 0.05 mg/mL CP III supplementation and/or irradiation with 450nm light at a radiant exposure of 7.56 J/cm²; average irradiance of 3mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 72 and 144 hours.

FIG. 36 is a graph depicting GBS survival for different combinations of0.002 or 0.02 mg/mL CP III supplementation and/or irradiation with 450nm light at a radiant exposure of 7.56 J/cm²; average irradiance of 3mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 72 and 144 hours.

FIGS. 37 and 38 are graphs depicting GBS survival for differentcombinations of 0.02 mg/mL PPIX supplementation (with optionalincubation and wash after supplementation) and/or irradiation with 450nm light at a radiant exposure of 7.56 J/cm²; average irradiance of 3mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 72 and 144 hours.

FIGS. 39, 40 and 41 are graphs depicting GBS survival for differentcombinations of 0.05 mg/mL PPIX supplementation (with optionalincubation and wash after supplementation) and/or irradiation with 450nm light at a radiant exposure of 7.56 J/cm²; average irradiance of 3mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 72 and 144 hours.

FIG. 42 is a graph depicting GBS survival for different combinations of0.05 mg/mL CP III supplementation (with optional incubation and washafter supplementation) and/or irradiation with 450 nm light at a radiantexposure of 7.56 J/cm²; average irradiance of 3 mW/cm²; pulsed mode ofirradiation (33% duty factor); and multiple irradiation at 0, 72 and 144hours.

FIGS. 43 and 44 are graphs depicting GBS survival for differentcombinations of 0.08 mg/mL PPIX supplementation (with optionalincubation and wash after supplementation) and/or irradiation with 450nm light at a radiant exposure of 7.56 J/cm²; average irradiance of 3mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 72 and 144 hours.

FIGS. 45, 46 and 47 are graphs depicting GBS survival for differentcombinations of 0.2 mg/mL PPIX supplementation (with optional incubationand wash after supplementation) and/or irradiation with 450 nm light ata radiant exposure of 7.56 J/cm²; average irradiance of 3 mW/cm²; pulsedmode of irradiation (33% duty factor); and multiple irradiation at 0, 72and 144 hours.

FIGS. 48 and 49 are graphs depicting GBS survival for differentcombinations of 0.2 mg/mL CP III supplementation (with optionalincubation and wash after supplementation) and/or irradiation with 450nm light at a radiant exposure of 7.56 J/cm²; average irradiance of 3mW/cm²; pulsed mode of irradiation (33% duty factor); and multipleirradiation at 0, 72 and 144 hours.

FIG. 50 is a graph depicting GBS survival for different combinations of0.08 mg/mL CP III supplementation (with optional incubation and washafter supplementation) and/or irradiation with 450 nm light at a radiantexposure of 7.56 J/cm²; average irradiance of 3 mW/cm²; pulsed mode ofirradiation (33% duty factor); and multiple irradiation at 0, 72 and 144hours.

FIG. 51 illustrates a light device incorporating printed light emittingdiodes placed externally on a cheek region of a human body for use inaccordance with the present invention.

FIG. 52 illustrates a light device incorporating printed light emittingdiodes placed externally on an inner wrist region of a human body foruse in accordance with the present invention.

FIGS. 53A and 53B illustrate a nasal applicator incorporating printedlight emitting diodes that is suitable for use in accordance with thepresent invention.

FIGS. 54A, 54B, 54C, 54D and 54E illustrate various embodiments of arespirator mask incorporating printed light emitting diodes that issuitable for use in accordance with the present invention.

FIG. 55 is a schematic illustration depicting an experimental set-upused in bacteria irradiation with pulsed 450 nm blue light.

FIG. 56 are light micrographs showing non-irradiated control MRSA (leftpanel) and irradiated MRSA (right panel).

FIG. 57 are transmission electron microscope images showing normal celldivision in non-irradiated control MRSA (A and C) and disruption ofnormal cell division in irradiated MRSA (B and D).

FIG. 58 are transmission electron microscope images showing structuraldamage to irradiated MRSA (B) compared to non-irradiated control MRSA(A), as well as normal cell division in non-irradiated control MRSA (A)compared to irradiated MRSA (B).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present invention is directed to methods and systems for thephotoeradication of microorganisms from a target using pulsed purple orblue light (and optionally pulsed ultraviolet, red or infrared light).While the invention will be described in detail below with reference tovarious exemplary embodiments, it should be understood that theinvention is not limited to the specific methodologies or deviceconfigurations of these embodiments. In addition, although the exemplaryembodiments are described as embodying several different inventivefeatures, one skilled in the art will appreciate that any one of thesefeatures could be implemented without the others in accordance with thepresent invention.

I. General Overview

The methods and systems of the present invention may be used tophotoeradicate many different types of microorganisms with pulsed purpleor blue light (and optionally pulsed ultraviolet, red or infrared light)provided at specified pulse parameters, dosages and time intervals. Insome embodiments, the pulsed light consists of a sequence of lightpulses each of which has a peak irradiance and a pulse durationsufficient to optically excite photoactive molecules capable ofphotoeradication of the microorganisms and move them to an excitedsinglet state. The light pulses are separated by an off time sufficientto allow the photoactive molecules to return to their ground state,which transition creates a reaction with triplet oxygen that reduces theoxygen molecule to a highly reactive singlet state which includessinglet oxygen (¹O₂), hydroxyl radicals (.OH) and superoxide (O₂ ⁻)ions. These are capable of destroying cellular structure of themicroorganisms and thereby photoeradicate all or a portion of themicroorganisms. This mechanism of microorganism suppression oreradication is broad spectrum and believed to be applicable to varioustypes of microorganisms, including bacteria, viruses and fungi. Incontrast, when light is applied in a continuous wave mode, thephotoactive molecules are maintained in an excited state and fewerreturn to a ground state (they do so randomly as opposed to a cascadethat follows the pulsing) and, thus, the effect of the light is reduced.

In a first embodiment, the invention is used to provide treatment formany different types of diseases, disorders, or conditions. For example,a region of skin, tissue, or a wound infected by bacteria may beirradiated with pulsed purple or blue light for the treatment of abacterial infection. The treatment can be provided to subjects having orwho are susceptible to or at elevated risk for experiencing a bacterialskin, tissue or wound infection. Subjects may be susceptible to or atelevated risk for experiencing a bacterial infection due to familyhistory, age, environment, and/or lifestyle. As used herein,“susceptible” and “at risk” refer to having little resistance to acertain disease, disorder or condition, including being geneticallypredisposed, having a family history of, and/or having symptoms of thedisease, disorder or condition.

The bacterial infection may comprise any infection caused by aerobic andanaerobic bacteria (Gram positive and Gram negative). Exemplarybacterial infections include acne, psoriasis, cellulitis, erysipelas,erythrasma, folliculitis and skin abscesses, hidradenitis suppurativa,impetigo and ecthyma, lymphadenitis, lymphangitis, necrotizing skininfections, staphylococcal scalded skin syndrome, as well as wound andtissue infections such as osteomyelitis.

As another example, cells infected by various types of viruses may beirradiated with pulsed purple or blue light for the treatment of a viralinfection. The treatment can be provided to subjects having or who aresusceptible to or at elevated risk for experiencing a viral infection.Exemplary viral infections include SARS-CoV, 2019-nCoV, humancoronavirus 229E, bat coronavirus HKU9-1, H1N1, norovirus (NoV) andfeline calicivirus (which are part of the Caliciviridae family),influenza virus type A, HCoV-OC43, and HCoV-NL63.

In particular, the current emergence of the 2019-nCoV and the previousemergence of the severe acute respiratory syndrome (SARS), caused by aformerly un-known coronavirus, SARS-CoV, exemplify the considerablezoonotic potential of coronaviruses and their ability to seriouslyaffect human health. The novel coronavirus, 2019-nCoV, is a virusclosely related to SARS-CoV and human coronavirus 229E, and originatedfrom Wuhan, China. Both 2019-nCoV and the SARS-CoV share a commonancestor that resembles the bat coronavirus HKU9-1.

The human airway epithelium (HAE) represents the entry port of manyhuman respiratory viruses, including coronaviruses (CoVs), which havevery similar spike protein 3-D structures that are considered to havestrong binding affinity to the human cell receptor,angiotensin-converting enzyme 2 (ACE2). Therefore, the cells with ACE2expression may act as target cells and thus are susceptible to 2019-nCoVinfection. Such cells include type II alveolar cells (AT2) orrespiratory epithelial cells of the lungs. The symptoms of COVID-19,which is the disease caused by 2019-nCoV, include fever, cough,shortness of breath, headache and sore throat. There are presently noantivirals or vaccines, and the current treatment of COVID-19 issupportive care (treatment of symptoms).

As another example, cells infected by various types of fungi may beirradiated with pulsed purple or blue light for the treatment of afungal infection. The treatment can be provided to subjects having orwho are susceptible to or at elevated risk for experiencing a fungalinfection. Exemplary fungal infections include tinea capitis, tineacorporis, tinea cruris, tinea pedis, and tinea unguium.

In a second embodiment, the invention is used to enable photoeradicationof microorganisms from contaminated environments. The microorganisms maycomprise any aerobic and anaerobic bacteria (Gram positive and Gramnegative) or any virus. For example, community associatedmethicillin-resistant Staphylococcus aureus (MRSA) strains or SARS-CoV,2019-nCoV, human coronavirus 229E, bat coronavirus HKU9-1, H1N1,norovirus (NoV) and feline calicivirus (which are part of theCaliciviridae family), influenza virus type A, HCoV-OC43, and HCoV-NL63are often acquired from places that people congregate, such as lockerrooms, public or private restrooms, airplanes, ambulances, fire trucks,emergency services personnel vehicles, schools, beaches, playgrounds,playing fields, etc. Hospital associated MRSA or SARS-CoV, 2019-nCoV,human coronavirus 229E, bat coronavirus HKU9-1, H1N1, norovirus (NoV)and feline calicivirus (which are part of the Caliciviridae family),influenza virus type A, HCoV-OC43, and HCoV-NL63 are typically contactedin hospitals and clinical environments. These contaminated environmentsmay be irradiated with pulsed purple or blue light to provide a moreeffective way to sanitize such places. The pulsed purple or blue lightmay be placed together with a white light to act as a dual light source,one for lighting and one for bactericidal or anti-viral effects. Thepulse treatment sequences could be applied daily to the environment oron any other periodic or scheduled basis (e.g., the pulse treatmentsequence could be applied to the interior surfaces of an airplanebetween the various successive flights).

In a third embodiment, the invention is used to enable photoeradicationof microorganisms from food. The bacteria may comprise any aerobic andanaerobic bacteria (Gram positive and Gram negative), norovirus (NoV)(which is part of the Caliciviridae family), or hepatitis A. Forexample, foods may be contaminated with Salmonella spp., E. Coli, orListeria spp. Salmonella spp. contains CspH, a cold shock protein thatprotects the bacteria at 50° C. As such, refrigeration at thistemperature may not be adequate to kill the bacteria. Refrigeration inretail may be at a higher temperature further exacerbating the problemof bacterial growth. Globally, there are more than 80.3 million casesannually of Salmonella spp. with more than 115,000 deaths. Contaminatedfoods may be irradiated with pulsed purple or blue light to provide amore effective way to extend shelf life and prevent bacterialtransmission to humans and animals exposed to such foods. The pulsedlight may be built into commercial, residential or portablerefrigeration systems, food display systems, food storage areas andsystems for food processing. For example, the pulsed purple or bluelight may be activated whenever a door of a refrigeration system isopened and closed. When the door is opened, the light would be white andwhen closed the pulsed purple or blue light would be applied at thecorrect dose and treatment sequence. The treatment sequence would beapplied daily and/or as new food items are added to the food storage orprocessing system.

For all three embodiments discussed above, exemplary bacteria includePropionibacterium acnes, Group B Streptococcus, Propionibacterium spp.,Staphylococcus spp. (including methicillin resistant strains),Clostridium spp., Escherichia spp., Pseudomonas spp., Campylocbacterspp., Listeria spp., Leuconostoc spp., Bacillus spp., Acinetobacterspp., Streptococcus spp., Brucella spp., Proteus spp., Klebsiella spp.,Shigella spp., Helicobacter spp., Mycobacterium spp., Enterococcus spp.,Salmonella spp., Chlamydia spp., Porphynomonas spp., Stenotrophomonasspp., and Elizabethkingia spp. Of course, other types of bacteria mayalso be targeted in accordance with the present invention.

Also, for all three embodiments discussed above, exemplary virusesinclude SARS-CoV, 2019-nCoV, human coronavirus 229E, bat coronavirusHKU9-1, H1N1, norovirus (NoV) and feline calicivirus (which are part ofthe Caliciviridae family), influenza virus type A, HCoV-OC43, HCoV-NL63,and hepatitis A.

In some cases, the microorganism itself synthesizes a photoactivemolecule that functions as a photoreceptor for the pulsed ultraviolet,purple or blue light—i.e., the photoactive molecule is endogenous to themicroorganisms. An example of this type of microorganism is thePropionibacterium acnes (P. acnes) bacteria, which synthesizes thephotoactive molecule porphyrin. In the case of viral microorganisms, thepulsed ultraviolet, purple or blue light may be absorbed by the viralRNA or protein photoreceptor leading to the destruction ordestabilization of the virus rendering it inactive.

In other cases, the microorganisms do not synthesize a sufficient amountof a photoactive molecule, such as the Group B Streptococcus (GBS)bacteria or different types of viruses, such as the coronavirus. Forexample, while GBS contains chromophores such as characteristicbrick-red pigment ornithine rhamnopolyenic (rhamnolipid), aβ-haemolysin/cytolysin, and granadaene, it is believed that thesechromophores are not implicated in the suppression of bacteria growthupon irradiation with pulsed blue or purple light. In these cases, themicroorganisms are associated or supplemented with a photoactivemolecule that is exogenous to the microorganisms and functions as aphotoreceptor for the pulsed purple or blue light, as discussed below.

In some embodiments, the microorganisms are suspended in a medium thatis endogenous to a human body (e.g., an organically-rick media (ORM)such as human saliva) and the medium contains the photoactivemolecule—i.e., the photoactive molecule is an extracellular endogenousphotosensitizer. For example, human saliva contains riboflavin,tyrosine, tryptophan, pyridoxine and folic acid.

In other embodiments, the microorganisms are hosted within a pluralityof cells that are endogenous to a human body and the cells themselvescontain the photoactive molecule. For example, it is believed that lungcells that have ACE2 expression, such as type II alveolar cells (AT2) orrespiratory epithelial cells, may be the main target cells during2019-nCoV infection. These cells may contain the photoactive moleculesporphyrin, NADH, and FAD. In the case of red blood cells which aretargets for viral invasion during 2019-nCoV infection, the blood cellcontains the photoactive molecule porphyrin.

In yet other embodiments, the photoactive molecule comprises aphotosensitizer that is administered to the target. Examples ofphotosensitizers that may be applied topically to skin, tissue or awound include curcumin, aminolevulinic acid (ALA), protoporphyrin IX(PPIX), coproporphyrin III (CP III), flavin mononucleotide (FMN), ornicotinamide adenine dinucleotide (NAD). These photosensitizers may beadministered in various doses that are typically in the range of 5 to 20micromolar. Of course, these doses may vary based on the photoactivationrequired and topical and systemic toxicity profiles. Examples ofphotosensitizers that may be ingested include curcumin, aminolevulinicacid (ALA), and chloroquine and its derivatives. These photosensitizersmay be administered in various doses from as little as 1 milligram to5000 milligrams per day using oral or intravenous administration. Ofcourse, these doses may vary based on the photoactivation required andsystemic toxicity profiles. It should be understood that thesephotosensitizers may be used in combination with any of the embodimentsdescribed above.

In accordance with the invention, bacteria, viruses or othermicroorganisms are inactivated via the application of ultraviolet,purple or blue light in a pulsed mode of irradiation. As used herein,“ultraviolet light” refers to light having a wavelength ranging fromabout 200 nm to about 380 nm (e.g., 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380 nm or somevalue therebetween). Particularly suitable wavelengths are about 260 nmand 370 nm. As used herein, “purple or blue light” refers to lighthaving a wavelength ranging from about 380 nm to about 560 nm (e.g.,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560 nm or some value therebetween). Particularlysuitable wavelength are about 410 nm and 450 nm.

Also, pulsed red light may be used for its anti-inflammatory effects toreduce the cytokine reaction response associated with the high levels ofReactive Oxygen Species (ROS) in targeted cells and tissues associatedwith microorganism destruction. As used herein, “red light” refers tolight having a wavelength ranging from about 600 nm to about 830 nm(e.g., 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830 nm or some valuetherebetween). Particularly suitable red wavelengths are about 640 nmand 645 nm. In addition, pulsed infrared light may be used to killmicroorganisms through the production and release of nitric oxide (NO)from the mitochondria. As used herein, “infrared light” refers to lighthaving a wavelength ranging from about 780 nm to about 1060 nm (e.g.,780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040,1050, 1060 nm or some value therebetween). Particularly suitableinfrared wavelengths are about 904 nm and 940 nm. The pulses providedcan be a square wave, rectified sinusoidal waveform or any combinationthereof, although other pulse shapes may also be used in accordance withthe present invention. A square wave with a rise time of less than 1microsecond is preferred.

The pulsed purple or blue light (and optionally pulsed ultraviolet, redor infrared light) is provided at radiant exposures that can range fromabout 0.5 J/cm² to about 60 J/cm², with a preferred range of about 3.6J/cm² to about 20 J/cm², and a more preferred range of about 3.6 J/cm²to about 5 J/cm². The average irradiance can range from about 0.1 mW/cm²to about 20 mW/cm², with an average irradiance of about 2 mW/cm² toabout 3 mW/cm² being preferred at the tissue surface, and may be higherfor contaminated environment or food surface.

It should be understood that the low levels of light used in connectionwith the present invention (e.g., average irradiances of about 2 mW/cm²to about 3 mW/cm² and radiant exposures of about 3.6 J/cm² to about 5J/cm²) are very safe to the eye and meet international blue light safetyrequirements, including IEC TR 62778:2014 (Application of IEC 62471 forthe assessment of blue light hazard to light sources and luminaries),IEC 62471:2006 (Photobiological safety of lamps and lamp systems), IEC60601-2-57:2011 (Medical Electrical equipment—Part 2-57: Particularrequirements for the basic safety and essential performance of non-laserlight source equipment intended for therapeutic, diagnostic, monitoringand cosmetic/aesthetic use). This provides a significant advantage overcontinuous wave (CW) light sources that deliver light at highirradiances and radiant exposures. These higher output light sourcesalso require significant cooling and heat sinking and, thus, are notideal for refrigeration applications.

The pulse parameters of the pulsed purple or blue light are preferablyselected to optimize the photochemical reaction of the photoactivemolecule used to photoeradicate the microorganisms, as described above.In most embodiments, the peak irradiance of the light pulses ranges from0.3 mW/cm² to 60 mW/cm² with a preferred range of about 6 mW/cm² toabout 15 mW/cm² at a duty factor of 33%. At other duty factors, the peakirradiance may be higher or lower so that the average irradiance stayswithin the preferred ranges of irradiance described herein. The dutyfactor is typically in the range of about 20% to about 33%.

In most embodiments, the pulse duration of the light pulses ranges from5 microseconds to 1,000 microseconds, and the off time between the lightpulses ranges from 10 microseconds to 1 second. A suitable combinationmay range from about 5 microseconds to about 30 microseconds for thepulse duration with off times ranging from about 10 microseconds toabout 100 microseconds. A particularly suitable combination provides apulse duration of about 10 microseconds and an off time of about 20microseconds. The light pulses are typically provided at a pulserepetition rate that ranges from 33 kHz to 40 kHz.

It should be understood that the optimal pulse parameters fordestruction of microorganisms will vary between different types ofmicroorganisms. For example, the pulse intensity and on/off states thatresult in the depletion and restoration/recovery of porphyrins or otherphotoactive molecules involved in bacterial photoeradication will varybetween different types of bacteria.

The pulsed purple or blue light (and optionally pulsed ultraviolet, redor infrared light) may be applied one time (i.e., single irradiationsession) and is preferably provided multiple times (i.e., multipleirradiation sessions) at the desired radiant exposures (fluence) andpower density (irradiance). One skilled in the art will appreciate thatthe irradiation time for each irradiation session is dependent on thedose, and is typically in the range of about 20 minutes to about 45minutes for the irradiances and radiant exposures used in connectionwith the present invention.

In some embodiments, the duration of exposure may be controlled by thedecay and recharge in fluorescence of the photoactive molecule. In thatconfiguration, the exposure would stop when the fluorescence hasdepleted to a preset level indicating that there is insufficientphoto-activity to maintain bacterial kill and then restart when thefluorescence has returned to another preset level. Also, the irradiationsessions may be timed to a replication cycle of the microorganisms sothat the irradiation sessions restart prior to replication of themicroorganisms.

Preferably, the pulsed purple or blue light (and optionally pulsedultraviolet, red or infrared light) is applied during a plurality ofirradiation sessions at pre-defined time intervals in accordance with anirradiation schedule, as described below. The time interval betweenirradiation sessions may range from about one hour to about four hours(e.g., 1, 2, 3, 4 hours or some value therebetween) with two or three(or more) irradiation sessions per day. These timed irradiation sessionsmay also be repeated on two, three, four or more days in certainembodiments, or in the case of environmental or food irradiation on adaily basis. In some embodiments, the pulsing has been found to beoptimized when the irradiation schedule includes the application ofpulses three times per day with a three hour time interval betweenapplications, which may be repeated on two or more days (e.g., when thepulses are applied at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours). Ofcourse, other irradiation schedules that are suitable for a particularapplication may also be used within the scope of the present invention.The irradiation schedule is preferably chosen to provide a survival ratefor the microorganisms of less than 50% and preferably 0% (i.e., asurvival rate of 50%, 40%, 30%, 20%, 10%, 5%, 0% or some valuetherebetween).

In addition to the basic pulsing of the purple or blue light asdescribed above, the pulsed mode may be further modulated when used forirradiation of human or animal tissue, i.e., gated on and off with a lowfrequency signal timed to coincide with the heart rate or at a ratesimilar to the heart rate (i.e., 0.5 to 2 Hz or a multiple or harmonicthereof). This signal allows for recharge of the free oxygen, porphyrinsor other molecules responsible for photochemical activation of cellularkilling mechanisms during the resting period of the photostimulation.Duty factors in the range of 5% to 95% may be used for this modulation.

Any suitable light source may be used to generate the pulsed purple orblue light (and optionally pulsed ultraviolet, red or infrared light) inaccordance with the present invention. Exemplary light sources includevarious types of lasers, light emitting diodes (LEDs), organic lightemitting diodes (OLEDs), printed light emitting diodes (printed LEDs),polymer light emitting diodes (PLEDs) also known as light emittingpolymers (LEPs), quantum dot light emitting diodes (QDLEDs), andfluorescent tubes emitting light in the purple or blue spectral region.Exemplary light sources are described in greater detail below.

For the first embodiment directed to the treatment of various diseases,disorders or conditions, devices comprised of printed LEDs or OLEDs onflexible substrates are preferred due to their ability to produce lightwith an intensity that is substantially constant across the surface ofthe light source so as to provide substantially uniform light emissionwhen the light source is in contact with or illuminates skin or tissueof a subject. The devices are also able to conform to the skin or tissuesurface to maximize optical coupling. In addition, a layer of hydrogelmay be used as an adhesive to contact the flexible substrate to the skinor tissue surface and thereby provide optical light piping and coupling.The hydrogel layer may also contain therapeutic substances used toenhanced bacterial inhibition or viral suppression or kill.

Various exemplary light devices that may be used to deliver pulsed lightin accordance with the invention are shown in FIGS. 51-54.

FIG. 51 illustrates a light device comprising an oblong-shaped lightsource 300 in the form of printed LEDs on a flexible substrate that iscontrolled by a controller 310, as described herein. In this embodiment,light source 300 is configured to be placed externally on a cheek regionof a human body. The pulsed light emitted by this device may comprisepulsed blue light, pulsed blue and red light, or pulsed blue andinfrared light and may be used, for example, to target microorganismssuspended in human saliva within the oral cavity. A biocompatiblehydrogel may also be used. In other embodiments, the light source isconfigured for internal placement within the oral cavity so as todirectly target the microorganisms suspended in human saliva.

FIG. 52 illustrates a light device comprising a butterfly-shaped lightsource 400 in the form of printed LEDs on a flexible substrate that iscontrolled by a controller 410, as described herein. In this embodiment,light source 400 is configured to be placed placed externally on aninner wrist region of a human body. The pulsed light emitted by thisdevice may comprise pulsed blue light, pulsed blue and red light, orpulsed blue and infrared light, for transcutaneous application over theinner wrist region (or other areas in close proximity to blood flow) inorder to target microorganisms in the blood, such as treatment of ablood borne virus on platelets or immune cells. A biocompatible hydrogelmay also be used.

FIGS. 53A and 53B illustrate a nasal applicator comprising headgear 500with a pair of nasal inserts 510 and 520 that are configured to beinserted into the nasal cavity, but allow breathing during use. Each ofnasal inserts 510 and 520 incorporates printed LEDs and may be used, forexample, to target microorganisms located within the nasal cavity. Thepulsed light emitted by this device may comprise pulsed blue light,pulsed blue and red light, or pulsed blue and infrared light, or acombination of all three wavelengths.

FIG. 54A illustrates a respirator mask that functions as part of an airfiltration system. The respirator mask includes an outer mask 600 thatsupports a light patch and filter assembly 610 configured to filterairborne particles (e.g., a bacteria or virus suspended in human saliva)from the air passed through various filter layers, as well asphotoeradicate all or a portion of the microorganisms (e.g., a bacteriaor virus) that become trapped within those filter layers therebypreventing the microorganisms from penetrating into the oral and nasalcavities and lungs of the user.

Light patch and filter assembly 610 is shown in greater detail in FIGS.54B and 54C and, in general, comprises a plurality of filter and lightlayers 612 sealed together by a sealing area 614. As best shown in FIG.54C, the layers of light patch and filter assembly 610 include an outerfilter layer 620 (i.e., the layer facing the environment), an outerfolded filter layer 622, a printed LED layer 624, an inner folded filterlayer 626, and an inner filter layer 628 (i.e., the layer facing theuser). Preferably, outer filter layer 620 and inner filter layer 628 areeach made of a material that is greater than 95% efficient at filtering0.3-μm particles (i.e., an N95 material), although other materials mayalso be used. Outer folded filter layer 622 and inner folded filterlayer 626 are each made of a filter material (including, but notlimited, to an N95 material) that is formed in a folded configuration,as shown, in order to provide support for printed LED layer 624 and toprovide a cavity that enables air circulation between outer and innerfilter layers 620 and 628 and printed LED layer 624, respectively. Inother embodiments, suitable spacers may be used in place of outer foldedfilter layer 622 and inner folded filter layer 626.

Printed LED layer 624 comprises printed LEDs on a flexible substrate, asdescribed herein, which is connected via a wire 618 to a controller thatincludes a power source, such as a rechargeable or non-rechargeablebattery pack (not shown). In this embodiment, printed LED layer 624 isconfigured to emit pulsed light in a direction toward outer foldedfilter layer 622 and outer filter layer 620. The pulsed light maycomprise a single wavelength or a combination of pulsed purple light,pulsed blue light or pulsed infrared light. The pulsed light is able tophotoeradicate all of a portion of the microorganisms (e.g., bacteria orviruses) that become trapped within outer folded filter layer 622 andouter filter layer 620—i.e., the pulsed light effectively “cleans” thosefilters. Preferably, outer folded filter layer 622 is made of a materialthat is semi-transparent so as to permit the transmission of pulsedlight from printed LED layer 624 to outer filter layer 620. In someembodiments, pulsed or continuous wave (CW) ultraviolet light (e.g.,UVA, UVB or UVC) may also be used for its bactericidal or anti-viraleffects.

In this embodiment, printed LED layer 624 comprises a solid layer(printed LEDs on a flexible substrate) that does not enable airflowtherethrough. Therefore, a pair of cutouts 616 a and 616 b are providedon each side of printed LED layer 624 to permit airflow around the sidesof printed LED layer 624. As such, the air flows transversely throughouter filter layer 620 and outer folded filter layer 622, laterally fromouter folded filter layer 622 around the sides of printed LED layer 624to inner folded filter layer 626, and then transversely through innerfolded filter layer 626 and inner filter layer 628. Non-breathablematerial 630 a and 630 b is provided on each side of inner filter layer628 to direct the air flow through this filter layer. It should be notedthat the filter layers are omitted in FIG. 54B in order to illustratethe position of cutouts 616 a and 616 b.

In other embodiments, the printed LED layer includes one or moreopenings that enable the direct flow of air from outer folded filterlayer 622 to inner folded filter layer 626. For example, FIG. 54Dillustrates a printed LED layer 642 and a sealing area 640 that aresimilar to those described in connection with FIG. 54B (noting againthat the filter layers are omitted in FIG. 54D), with the exception thatprinted LED layer 642 includes a plurality of vertical slots 644 toenable the flow of air therethrough. As another example, FIG. 54Eillustrates a printed LED layer 652 and a sealing area 650 that aresimilar to those described in connection with FIG. 54B (noting againthat the filter layers are omitted in FIG. 54E), with the exception thatprinted LED layer 652 includes a plurality of holes 654 to enable theflow of air therethrough. Of course, the number and shape of theopenings in the printed LED layer will vary between differentembodiments. It should be understood that cutouts 616 a and 616 b arenot required in these embodiments.

In some embodiments, printed LED layer 624 (or the printed LED layersshown in FIGS. 54D and 54E) include LEDs printed on both sides of thesubstrate so that pulsed light is also emitted in a direction towardinner folded filter layer 626 and inner filter layer 628. The pulsedlight may comprise a single wavelength or a combination of pulsed purplelight, pulsed blue light or pulsed infrared light. The pulsed light isable to photoeradicate all of a portion of the microorganisms (e.g.,bacteria or viruses) that become trapped within inner folded filterlayer 626 and inner filter layer 628—i.e., the pulsed light effectively“cleans” those filters. Preferably, inner folded filter layer 626 ismade of a material that is semi-transparent so as to permit thetransmission of pulsed light from printed LED layer 624 to inner filterlayer 628. In some embodiments, pulsed or continuous wave (CW)ultraviolet light (e.g., UVA, UVB or UVC) may also be used for itsbactericidal or anti-viral effects.

In yet other embodiments, a second printed LED layer (not shown) ispositioned between the user and inner filter layer 628 and configured toemit pulsed light in a direction toward inner filter layer 628 and innerfolded filter layer 626. The pulsed light may comprise a singlewavelength or a combination of pulsed purple light, pulsed blue light orpulsed infrared light. The pulsed light is able to photoeradicate all ofa portion of the microorganisms (e.g., bacteria or viruses) that becometrapped within inner filter layer 628 and inner folded filter layer626—i.e., the pulsed light effectively “cleans” those filters.Preferably, inner filter layer 628 is made of a material that issemi-transparent so as to permit the transmission of pulsed light fromthe second printed LED layer to inner folded filter layer 626. In someembodiments, pulsed or continuous wave (CW) ultraviolet light (e.g.,UVA, UVB or UVC) may also be used for its bactericidal or anti-viraleffects.

It should be understood that the present invention is not limited to theconfiguration of the respirator mask described above, and that othermask designs may also be used within the scope of the invention. Forexample, in some embodiments, the printed LED layer is removeablysecured within the respirator mask in such a manner as to enable accessto the printed LED layer. For example, the printed LED layer may bepositioned within a compartment that is closeable using Velcro® or anyother suitable fastener, but which enables access to the compartment forremoval of the printed LED layer. This configuration enables the printedLED layer to be inserted into a variety of different types of respiratormasks. Thus, while the respirator mask itself is disposable, the printedLED layer is re-useable in different masks following cleaning anddecontamination. Also, other types of light sources may also be used inaccordance with the present invention.

Any of the light devices described above may also be used in combinationwith detectors to verify the kill rates or to filter change points.

For the second and third embodiments directed to photoeradication ofmicroorganisms from contaminated environments and food, respectively, itis possible to use strips or sheets of printed LEDs or OLEDs, high powerLEDs or even tube lamps emitting modulated light in the UVA through bluespectral range. Certain of these strips or sheets may have rows of bluelamps in parallel or interspersed with white lamps or phosphor coated orStokes conversion quantum dots or the like to produce white light. Asused herein, “white light” means light having a color temperature in therange of about 1700 kelvins (K) to about 9500 kelvins (K).

In some embodiments, the light source is controlled by an electroniccircuit with a drive circuit and microcontroller that provides apreprogrammed sequence of light pulses at a fixed dose during eachirradiation session. In other embodiments, the light source iscontrolled by an electronic circuit with one or more sensors thatoperate in a closed loop to provide feedback to a microcontroller so asto dynamically control the dose during a treatment session. Exemplaryelectronic circuits for both preprogrammed control and dynamic controlof a light source are described in greater detail below. In someembodiments, the controller will receive input from a lighting system orthe opening or closing of a door in a refrigeration or storage unit toactivate the pulsed purple or blue lights and provide the appropriatedaily irradiation sequence.

II. In Vitro Testing of P. acnes Cultures to Determine Feasibility andReproducibility of Inactivation with Light Irradiation

Testing was performed to determine the feasibility and reproducibilityof 450 nm light to inactivate P. acnes using printed LED flexible lampsoperated in a continuous wave (CW) irradiation mode, a pulsedirradiation mode with a 33% duty factor, and a pulsed irradiation modewith a 20% duty factor (referred to hereinafter as CW mode, 33% DFpulsed mode, and 20% DF pulsed mode).

Culturing P. acnes

A vial of P. acnes (ATCC 6919) was obtained from the American TypeCulture Collection (ATCC) and cultures started according to ATCCrecommendations. Briefly, under anaerobic conditions, the lyophilizedpellet was rehydrated in 500 μL modified reinforced clostridial broth(pre-reduced) and transferred into another tube containing 5 mLclostridial broth. Test tubes were labelled as “Start Cultures” (SC) and200 μL transferred onto a reinforced clostridial agar plate. Thebacteria were streaked on the plates for isolation of single colonies bythe “clock plate technique” to check colony morphology and purity. Theseplates were called “start plates.” Both tube/plate were placed in ananaerobic chamber with BD Gas-Pak EZ anaerobic container system sachets.The anaerobic chamber was then placed inside a 37° C. incubator for 72hours. After 72 hours of growth period, culture tubes were removed fromthe 37° C. incubator and tested for their susceptibility to irradiationwith 450 nm blue light.

Illumination of Bacteria

A 5 mL liquid culture was grown for 3 days and 1 mL pipetted into asterile microcentrifuge tube and centrifuged at 13,300 rpm for fiveminutes. The supernatant was removed and discarded. The pellet wasre-suspended in 1 mL saline and optical density adjusted using McFarlandstandard to 0.8 to 1.0 at 625 nm for a concentration of 10⁸ CFU/mL. Thebacteria were diluted to a concentration of 1×10⁶ CFU/mL and 2 μLstreaked onto reduced clostridial agar plates (12 well plates were used,which enabled triplicate irradiations with 3 patches simultaneously) andirradiated in an anaerobic chamber with various protocols. A set ofnon-irradiated plates served as controls. After irradiation, plates wereplaced upside down in the anaerobic chamber, along with a Gas-pak sachetand incubated at 37° C. for 72 hours. The colonies were then counted,percentage survival computed and morphology checked. Illumination timesand intervals were optimized based on experimentation as to which doseand sequence provided the most effective kill rates.

Quantification of Bacterial Colonies

Standardized digital images of P. acnes colonies were taken 72 hoursafter incubation, with the camera positioned 10 cm perpendicularly aboveeach plate to ensure consistency of colony magnification. Colonies werecounted, and percent survival calculated, comparing irradiated groupsand non-irradiated controls.

Description of Test Protocols and Test Results Example 1

A first series of experiments involved culturing P. acnes andilluminating the bacteria with a 450 nm lighted substrate, an example ofwhich is shown in FIG. 22 (described below). The lighted substrate wasset to operate in CW mode and driven with a constant current source. Thepeak and average irradiances were the same, i.e., 4.5 mW/cm², due to thecontinuous output. The bacteria were irradiated one, three or four timeswith different radiant exposures, as described below. Three substrateswere provided for the experiments and all were tested and calibrated.

The data is presented in FIGS. 1-4. As illustrated in FIG. 1, singleirradiation of bacteria with radiant exposures of 20 J/cm² and 60 J/cm²produced statistically significant decreases in percent survival whencompared to the control, with maximum reduction of 31% observed at 60J/cm². As illustrated in FIG. 2, triple irradiation of bacteria with aradiant exposure of 20 J/cm² at 0, 24 and 48 hours reduced the percentsurvival to 62%, which was significantly different from the control. Asillustrated in FIG. 3, when bacteria were irradiated four times with aradiant exposure of 20 J/cm² at 0, 4, 24 and 48 hours, a significantdecrease in percent survival of 31% was observed when compared to thecontrol. This percent survival was much lower than the percent survivalof 62% observed with triple irradiation at 0, 24 and 48 hours (FIG. 2).It was found that four exposures at 20 J/cm² applied at 0, 4, 24 and 48hours provided the highest kill rates as compared to a single, double ortriple exposure.

FIG. 4 illustrates the relationship between different radiant exposuresfor a double exposure occurring at 0 and 4 hours. Double irradiation ofbacteria with radiant exposures of 30, 40, 45 and 60 J/cm² at 0 and 4hours produced a significant dose dependent decrease in percent survivalas the dose increased, with complete inactivation observed at 60 J/cm².

Example 2

A second series of experiments involved culturing P. acnes andilluminating the bacteria with the 450 nm lighted substrate shown inFIG. 22. The lighted substrate was set to operate in CW mode, 33% DFpulsed mode, or 20% DF pulsed mode, as described below, and driven witha constant current source. The average irradiance was the same for allmodes, i.e., 5 mW/cm². The peak irradiance was 15 mW/cm² in the 33% DFpulsed mode (i.e., three times higher than CW mode) and 25 mW/cm² in the20% DF pulsed mode (i.e., five times higher than CW mode). In the 33% DFpulsed mode, the pulse duration was 10 microseconds with an off time of20 microseconds, and the pulse repetition rate was 33 kHz. In the 20% DFpulsed mode, the pulse duration was 5 microseconds with an off time of20 microseconds, and the pulse repetition rate was 40 kHz. The bacteriawere irradiated twice at 0 and 4 hours with different radiant exposures,as described below. Three substrates were provided for the experimentsand all were tested and calibrated.

The data is presented in FIGS. 5 and 6. As illustrated in FIG. 5, doubleirradiation of bacteria at 0 and 4 hours with a radiant exposure of 60J/cm² in CW mode and 33% DF pulsed mode revealed marked decreases inpercent survival when compared to the control, with maximum reduction toa percent survival of 2.2% observed in the 33% DF pulsed mode. Asillustrated in FIG. 6, double irradiation of bacteria at 0 and 4 hourswith a radiant exposure of 20 J/cm² in CW mode, 33% DF pulsed mode and20% DF pulsed mode again revealed that the 33% DF pulsed mode was moreefficacious in suppressing bacteria growth than the CW mode and the 20%DF pulsed mode, with a percent survival of 16% for the 33% DF pulsedmode compared to percent survivals of 87% and 80% for the CW mode and20% DF pulsed mode, respectively.

Example 3

A third series of experiments involved further testing with a change tothe exposure schedule based on the findings described in FIG. 3, i.e.,that four irradiation exposures (0, 4, 24 and 48 hours) provided thebest kill rate at a radiant exposure of 20 J/cm². These experimentsinvolved culturing P. acnes and illuminating the bacteria with the 450nm lighted substrate shown in FIG. 22. The lighted substrate was set tooperate in CW mode or 33% DF pulsed mode, as described below, and drivenwith a constant current source. The average irradiance was selectable at2 and 3.5 mW/cm² in the 33% DF pulsed mode compared to an averageirradiance of 5 mW/cm² in the CW mode. In the 33% DF pulsed mode, thepulse duration was 10 microseconds with an off time of 20 microseconds,and a pulse repetition rate of 33 kHz. The bacteria were irradiated fourtimes at 0, 4, 24 and 48 hours with different radiant exposures of 5, 10and 20 J/cm². Three substrates were provided for the experiments and allwere tested and calibrated.

The data is presented in FIGS. 7-9. As illustrated in FIG. 7,irradiation of cultures four times (0, 4, 24 and 48 hours) with radiantexposures of 5, 10 and 20 J/cm² and an average irradiance of 2 mW/cm² inthe 33% DF pulsed mode showed significant decreases in percent survivalwhen compared to the control; however, no significant differences wereobserved between the doses. These results indicated that no significantdifferences in the percent survival existed with increases in radiantexposure from 5 J/cm² to 20 J/cm² at an average irradiance of 2 mW/cm².

As illustrated in FIG. 8, irradiation of cultures four times (0, 4, 24and 48 hours) with radiant exposures of 5, 10 and 20 J/cm² and anaverage irradiance of 3.5 mW/cm² in the 33% DF pulsed mode showedsignificant decreases in percent survival when compared to the control;however, no significant differences were observed between the doses.These results indicate that no significant differences in the percentsurvival existed with increases in radiant exposure from 5 J/cm² to 20J/cm² at an average irradiance of 3.5 mW/cm².

As illustrated in FIG. 9, irradiation of cultures four times (0, 4, 24and 48 hours) with radiant exposures 5, 10 and 20 J/cm² and anirradiance of 5 mW/cm² in the CW mode showed significant decreases inpercent survival when compared to the control. However, unlike theresults shown in FIGS. 7 and 8, the decrease in percent bacterialsurvival was dose dependent with significant decreases observed betweenthe radiant exposures of 5 J/cm² and 10 J/cm² and between 10 J/cm² and20 J/cm² at an irradiance of 5 mW/cm².

Thus, it can be seen that the CW mode behaved in a linear dose dependentfashion. However, a significant and unexpected result was obtained withrespect to the 33% DF pulsed mode, i.e., there was no significantradiant exposure dependency on kill rates for different averageirradiances of 2 and 3.5 mW/cm². As a result, a novel treatment wasdiscovered using two to four doses in sequence in the 33% DF pulsed modeat a radiant exposure of 5 J/cm².

III. In Vitro Testing of P. acnes Cultures to Determine OptimalParameters of Inactivation with Light Irradiation

The preliminary results described above revealed that pulsed blue lightwas more effective in suppressing bacteria growth than continuous wave.Thus, further testing was performed to determine the optimal parametersof 450 nm light to inactivate P. acnes using printed LED flexible lampsoperated in a pulsed irradiation mode with a 33% duty factor.

A suspension of P. acnes bacteria diluted to a concentration of 1×10⁶CFU/mL, as discussed in Section II above, was streaked onto reducedclostridial agar plates that were irradiated or not (controls) in ananaerobic chamber. The tests were conducted using three differentirradiation substrates (with the same power output) with each placed atthe top of two wells on a 12 well plate. This enabled results to beobtained in triplicates (six sets of data for each experiment). Afterirradiation protocols were completed, plates were placed upside down inthe anaerobic chamber, along with a Gas-pak sachet and incubated at 37°C. for 72 hours. The colonies were then counted, percentage survivalcomputed and morphology checked.

Description of Test Protocols and Test Results Example 1

A first experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 3mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated four times at 0, 4, 24 and 48 hours with different radiantexposures of 5, 10 and 20 J/cm².

As illustrated in FIG. 10, irradiation of cultures four times (0, 4, 24and 48 hours) with radiant exposures of 5, 10 and 20 J/cm² and anaverage irradiance of 3 mW/cm² in the 33% DF pulsed mode showeddecreases in percent survival when compared to the control, i.e., 89.1%,34.3% and 0.5%, respectively. The decrease in percent bacterial survivalwas dose dependent with significant decreases observed between theradiant exposure of 5 and 10 J/cm², between 5 and 20 J/cm², and between10 and 20 J/cm².

Example 2

A second experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 3mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated four times at 0, 3, 6 and 24 hours with different radiantexposures of 5, 10 and 20 J/cm².

As illustrated in FIG. 11, irradiation of cultures four times (0, 3, 6and 24 hours) with radiant exposures of 5, 10 and 20 J/cm² and anaverage irradiance of 3 mW/cm² in the 33% DF pulsed mode showeddecreases in percent survival when compared to the control, i.e., 85.7%,16.3% and 0.0%, respectively. The overall decrease in percent survivalwas higher than that seen in the first example. Again, the decrease inpercent bacterial survival was dose dependent with significant decreasesobserved between the radiant exposure of 5 and 10 J/cm², between 5 and20 J/cm², and between 10 and 20 J/cm².

Example 3

A third experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 3mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated three times at 0, 3 and 6 hours with different radiantexposures of 5, 10 and 20 J/cm².

As illustrated in FIG. 12, irradiation of cultures three times using ashorter time interval (0, 3 and 6 hours) with radiant exposures of 5, 10and 20 J/cm² and an average irradiance of 3 mW/cm² in the 33% DF pulsedmode showed decreases in percent survival when compared to the control,i.e., 60.9%, 11.7% and 0.2%, respectively. The decrease in percentbacterial survival was dose dependent with significant decreasesobserved between the radiant exposure of 5 and 10 J/cm², between 5 and20 J/cm², and between 10 and 20 J/cm².

Example 4

A fourth experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 3mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated nine times at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours with aradiant exposure of 5 J/cm².

As illustrated in FIG. 13, a change in the irradiation schedule to threetimes per day, every three hours over the course of three days for atotal of nine times (0, 3, 6, 24, 27, 30, 48, 51 and 54 hours) with aradiant exposure of 5 J/cm² and an average irradiance of 3 mW/cm² in the33% DF pulsed mode showed a significant decrease in percent survivalwhen compared to the control, i.e., 2.57%.

Example 5

A fifth experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 3mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated twelve times at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and78 hours with a radiant exposure of 5 J/cm².

As illustrated in FIG. 14, a change in the irradiation schedule to threetimes per day, every three hours over the course of four days for atotal of twelve times (0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78hours) with a radiant exposure of 5 J/cm² and an average irradiance of 3mW/cm² in the 33% DF pulsed mode showed a significant decrease inpercent survival when compared to the control, i.e., 1.13%. The overalldecrease in percent survival was higher than that seen in the fourthexample when bacteria were irradiated over the course of three days(with all other parameters being the same).

Example 6

A sixth experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 2mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated twelve times at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and78 hours with a radiant exposure of 5 J/cm².

As illustrated in FIG. 15, the irradiation of bacteria three times perday, every three hours over the course of four days for a total oftwelve times (0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78 hours) witha radiant exposure of 5 J/cm² and a change in the average irradiance to2 mW/cm² in the 33% DF pulsed mode showed a complete bacterialeradication when compared to the control, i.e., 0.0%. The overalldecrease in percent survival was slightly higher than that seen in thefifth example when the average irradiance was 3 mW/cm² (with all otherparameters being the same).

Example 7

A seventh experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 2mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated nine times at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours with aradiant exposure of 5 J/cm².

As illustrated in FIG. 16, the irradiation of bacteria three times perday, every three hours over the course of three days for a total of ninetimes (0, 3, 6, 24, 27, 30, 48, 51 and 54 hours) with a radiant exposureof 5 J/cm² and an average irradiance of 2 mW/cm² in the 33% DF pulsedmode showed a complete bacterial eradication when compared to thecontrol, i.e., 0.0%. The overall decrease in percent survival was thesame as that seen in the sixth example when the irradiation was appliedover the course of four days for a total of twelve times (with all otherparameters being the same).

Example 8

An eighth experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 2mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated eight times at 0, 3, 24, 27, 48, 51, 72 and 75 hours with aradiant exposure of 3.6 J/cm².

As illustrated in FIG. 17, the irradiation of bacteria two times perday, every three hours over the course of four days for a total of eighttimes (0, 3, 24, 27, 48, 51, 72 and 75 hours) with a lower radiantexposure of 3.6 J/cm² and an average irradiance of 2 mW/cm² in the 33%DF pulsed mode showed a slight decrease in percent survival whencompared to the control, i.e., 86.9%, which is significantly higher thanthat seen in the seventh example when the radiant exposure was 5 J/cm²and the irradiation was applied three times per day (with all otherparameters being the same).

Example 9

A ninth experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 2mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated ten times at 0, 3, 24, 27, 48, 51, 72, 75, 96, and 99 hourswith a radiant exposure of 3.6 J/cm².

As illustrated in FIG. 18, the irradiation of bacteria two times perday, every three hours over the course of five days for a total of tentimes (0, 3, 24, 27, 48, 51, 72, 75, 96, and 99 hours) with a radiantexposure of 3.6 J/cm² and an average irradiance of 2 mW/cm² in the 33%DF pulsed mode showed a decrease in percent survival when compared tothe control, i.e., 78.9%, which is just slightly lower than that seen inthe eighth example when the irradiation was applied over the course offour days (with all other parameters being the same).

Example 10

A tenth experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 2mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated twelve times at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and78 hours with a radiant exposure of 3.6 J/cm².

As illustrated in FIG. 19, the irradiation of bacteria three times perday, every three hours over the course of four days for a total oftwelve times (0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78 hours) witha radiant exposure of 3.6 J/cm² and an average irradiance of 2 mW/cm² inthe 33% DF pulsed mode showed a decrease in percent survival whencompared to the control, i.e., 32.6%, which is lower than that seen inthe eighth and ninth examples.

Example 11

An eleventh experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 2mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated nine times at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours with aradiant exposure of 5 J/cm².

As illustrated in FIG. 20, the irradiation of bacteria three times perday, every three hours over the course of three days for a total of ninetimes (0, 3, 6, 24, 27, 30, 48, 51 and 54 hours) with a radiant exposureof 5 J/cm² and an average irradiance of 2 mW/cm² in the 33% DF pulsedmode showed a complete bacterial eradication when compared to thecontrol, i.e., 0.0%. This experiment confirmed the data from the seventhexperiment (see Example 7).

Example 12

A twelfth experiment involved culturing P. acnes and illuminating thebacteria with the 450 nm lighted substrate shown in FIG. 22. The lightedsubstrate was set to operate in 33% DF pulsed mode, as described below,and driven with a constant current source. The average irradiance was 2mW/cm². The pulse duration was 10 microseconds with an off time of 20microseconds, and a pulse repetition rate of 33 kHz. The bacteria wereirradiated nine times at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours with aradiant exposure of 3.6 J/cm².

As illustrated in FIG. 21, the irradiation of bacteria three times perday, every three hours over the course of three days for a total of ninetimes (0, 3, 6, 24, 27, 30, 48, 51 and 54 hours) with a radiant exposureof 3.6 J/cm² and an average irradiance of 2 mW/cm² in the 33% DF pulsedmode showed a significant decrease in percent survival when compared tothe control, i.e., 11.7%. Although the percentage survival was low, itwas still higher than that seen in the eleventh example when the radiantexposure was 5 J/cm² (with all other parameters being the same).

Summary

Further testing was performed using light patches that emit pulsed bluelight at a 33% duty cycle. The various protocols tested showed effectivereduction in bacteria growth. Testing using irradiation intervals of 3hours proved to be more efficient than irradiation intervals of 4 hours.It is believed that such observed efficiency may be due in part totargeting the bacteria at the appropriate time during the replicationcycle or due to porphyrin depletion/replenishing mechanisms in thebacteria.

Irradiation protocols were optimized to yield 100% bacterial suppressionof 1×10⁶ CFU/mL P. acnes cultures. Optimal bacterial suppression of 100%was attained (i) when cultures were irradiated at 0, 3, 6 and 24 hourswith a radiant exposure of 20 J/cm² and an average irradiance of 3mW/cm² (see Example 2), (ii) when cultures were irradiated three timesper day, every three hours over the course of four days for a total oftwelve times (0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78 hours) witha radiant exposure of 5 J/cm² and an average irradiance to 2 mW/cm² (seeExample 6), and (iii) when cultures were irradiated three times per day,every three hours over the course of three days for a total of ninetimes (0, 3, 6, 24, 27, 30, 48, 51 and 54 hours) with a radiant exposureof 5 J/cm² and an average irradiance of 2 mW/cm² (see Example 11).

One skilled in the art will appreciate that the present invention allowsfor improved device performance with reduced battery requirements, asubstantial reduction in treatment time, and improved patient safety dueto a significant reduction in heat and optical hazard.

IV. In Vitro Testing of GBS Cultures Supplemented with Porphyrins toObtain Bacterial Suppression with Light Irradiation

The results described above revealed that pulsed blue light waseffective in suppressing bacteria growth for P. acnes, which synthesizesthe photoactive molecule porphyrin. However, some bacteria such as GroupB Streptococcus (GBS) do not synthesize a sufficient amount of porphyrinor other photoactive molecules, in which case a photosensitizer isneeded to function as a photoreceptor for the pulsed blue light. Thus,further testing was performed to assess GBS bacterial suppression whensupplemented with exogenous porphyrins (either protoporphyrin IX (PPIX)or coproporphyrin III (CP III)) and irradiated with pulsed blue lightusing printed LED flexible lamps.

Study 1: GBS Supplemented With Different Concentrations of Porphyrins

GBS wild type strain COH1 was added to 3 mL of Todd Hewitt broth andgrown aerobically overnight at 37° C. From the overnight cultures, 500uL of GBS was added to fresh Todd Hewitt broth and grown logarithmicallyat 37° C. until reaching a concentration of 10⁸ CFU/mL. The culture wasthen centrifuged at 1300 rpm for three minutes, the supernatant wasremoved and discarded, and 1 mL of fresh Todd Hewitt broth was added.Serial dilutions of NaCl were made to obtain a suspension of GBS dilutedto a concentration of 1×10⁴ CFU/mL. The GBS were streaked onto ToddHewitt agar plates both with and without the addition of dilutedporphyrins (different concentrations of PPIX or CP III).

Some of the plates were irradiated in a chamber using the 450 nm lightedsubstrate shown in FIG. 22. The lighted substrate was set to operate in33% DF pulsed mode and driven with a constant current source. Theaverage irradiance was 3 mW/cm². The pulse duration was 10 microsecondswith an off time of 20 microseconds, and a pulse repetition rate of 33kHz. The plates subject to light treatment were irradiated three timesat 0, 72 and 144 hours with a radiant exposure of 7.56 J/cm² during eachirradiation session (i.e., each irradiation session was 42 minutes withan incubation period there between of 30 minutes).

After irradiation protocols were completed, the plates were placedupside down in the anaerobic chamber and incubated at 37° C. for 24hours. The plates were photographed, colonies counted and statistics ofpercent survival recorded.

Example 1

In a first experiment, the GBS were assigned to six experimental groups:(1) GBS only (control); (2) GBS and light; (3) GBS supplemented with0.05 mg/mL PPIX; (4) GBS supplemented with 0.002 mg/mL PPIX and light;(5) GBS supplemented with 0.02 mg/mL PPIX and light; and (6) GBSsupplemented with 0.05 mg/mL PPIX and light. The results are shown inFIG. 33.

As can be seen in FIG. 33, irradiation of GBS without the addition ofany PPIX showed a minimal decrease in percent GBS survival when comparedto the control, i.e., approximately 87%, which demonstrates that GBSdoes not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.05 mg/mL PPIX, but without any irradiation, did notshow much decrease in percent GBS survival when compared to the control,i.e., approximately 85%. In addition, irradiation of GBS supplementedwith 0.002 mg/mL PPIX did not show much decrease in percent GBS survivalwhen compared to the control, i.e., approximately 83%, whichdemonstrates that the concentration of PPIX was too low. However,irradiation of GBS supplemented with 0.02 mg/mL PPIX and 0.05 mg/mL PPIXshowed significant decreases in percent GBS survival when compared tothe control, i.e., 0% and 3%, respectively. Thus, the decrease inpercent GBS survival was dependent on the concentration of PPIX added tothe GBS prior to irradiation.

Example 2

In a second experiment conducted separately, and with observed slightdecrease in CFU/mL, the GBS were assigned to six experimental groups:(1) GBS only (control); (2) GBS and light; (3) GBS supplemented with0.05 mg/mL PPIX; (4) GBS supplemented with 0.002 mg/mL PPIX and light;(5) GBS supplemented with 0.02 mg/mL PPIX and light; and (6) GBSsupplemented with 0.05 mg/mL PPIX and light. The results are shown inFIG. 34.

As can be seen in FIG. 34, irradiation of GBS without the addition ofany PPIX showed a small decrease in percent GBS survival when comparedto the control, i.e., approximately 81%, which again demonstrates thatGBS does not synthesize a sufficient amount of porphyrin. GBSsupplemented with 0.05 mg/mL PPIX, but without any irradiation, showedsome decrease in percent GBS survival when compared to the control,i.e., approximately 50%. In addition, irradiation of GBS supplementedwith 0.002 mg/mL PPIX showed even more decrease in percent GBS survivalwhen compared to the control, i.e., approximately 37%. Further,irradiation of GBS supplemented with 0.02 mg/mL PPIX and 0.05 mg/mL PPIXshowed complete bacterial kill, i.e., 0% GBS survival. Thus, thedecrease in percent GBS survival was dependent on the concentration ofPPIX added to the GBS prior to irradiation, and also on the number ofCFU/mL.

Example 3

In a third experiment, the GBS were assigned to five experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.05 mg/mL CP III; (4) GBS supplemented with 0.002 mg/mL CP III andlight; and (5) GBS supplemented with 0.05 mg/mL CP III and light.Because the CP III was solubilized in ethanol, two additionalexperimental groups were included to test whether the quantity ofethanol used as a solvent had any significant effect on bacterial growthsuppression: (6) ethanol only and (7) ethanol and light. The results areshown in FIG. 35.

As can be seen in FIG. 35, irradiation of GBS without the addition ofany CP III showed a minimal decrease in percent GBS survival whencompared to the control, i.e., approximately 98%, which demonstratesthat GBS does not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.05 mg/mL CP III, but without any irradiation, didnot show much decrease in percent GBS survival when compared to thecontrol, i.e., approximately 84%. In addition, irradiation of GBSsupplemented with 0.002 mg/mL CP III did not show much decrease inpercent GBS survival when compared to the control, i.e., approximately78%, which demonstrates that the concentration of CP III was too low.However, irradiation of GBS supplemented with 0.05 mg/mL CP III showedcomplete bacterial kill, i.e., 0% GBS survival. Thus, the decrease inpercent GBS survival was dependent on the concentration of CP III addedto the GBS prior to irradiation. It should also be noted that ethanoldid not have any significant contribution to the observed suppressiveeffect.

Example 4

In a fourth experiment, the GBS were assigned to five experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.02 mg/mL CP III; (4) GBS supplemented with 0.002 mg/mL CP III andlight; and (5) GBS supplemented with 0.05 mg/mL CP III and light.Because the CP III was solubilized in ethanol, two additionalexperimental groups were included to test whether the quantity ofethanol used as a solvent had any significant effect on bacterial growthsuppression: (6) ethanol only and (7) ethanol and light. The results areshown in FIG. 36.

As can be seen in FIG. 36, irradiation of GBS without the addition ofany CP III showed a minimal decrease in percent GBS survival whencompared to the control, i.e., approximately 90%, which againdemonstrates that GBS does not synthesize a sufficient amount ofporphyrin. Also, GBS supplemented with 0.02 mg/mL CP III, but withoutany irradiation, showed a small decrease in percent GBS survival whencompared to the control, i.e., approximately 74%. In addition,irradiation of GBS supplemented with 0.002 mg/mL CP III showed asignificant decrease in percent GBS survival when compared to thecontrol, i.e., approximately 17%. Further, irradiation of GBSsupplemented with 0.05 mg/mL CP III showed complete bacterial kill,i.e., 0% GBS survival. Thus, the decrease in percent GBS survival wasdependent on the concentration of CP III added to the GBS prior toirradiation, and also on the number of CFU/mL. It should also be notedthat ethanol did not have any significant contribution to the observedsuppressive effect.

Study 2: GBS Supplemented with Porphyrins with Incubation afterSupplementation

GBS wild type strain COH1 was added to 3 mL of Todd Hewitt broth andgrown aerobically overnight at 37° C. From the overnight cultures, 500uL of GBS was added to fresh Todd Hewitt broth and grown logarithmicallyat 37° C. until reaching a concentration of 108 CFU/mL. The culture wasthen centrifuged at 1300 rpm for three minutes, the supernatant wasremoved and discarded, and 1 mL of fresh Todd Hewitt broth was added.

The culture was then split into three Eppendorf tubes—a first tube forGBS and second and third tubes for GBS with diluted porphyrins used forincubation. Diluted porphyrins (PPIX or CP III) were added into thesecond and third tubes and incubated for 30 minutes at 37° C. The thirdtube was then washed three times with phosphate-buffered saline (PBS)and centrifuged at 1300 rpm for three minutes, and the supernatant wasremoved and discarded. Serial dilutions of NaCl were made to obtain asuspension of GBS diluted to a concentration of 1×10⁴ CFU/mL. The GBSwere streaked onto Todd Hewitt agar plates.

Some of the plates were irradiated in a chamber using the 450 nm lightedsubstrate shown in FIG. 22. The lighted substrate was set to operate in33% DF pulsed mode and driven with a constant current source. Theaverage irradiance was 3 mW/cm². The pulse duration was 10 microsecondswith an off time of 20 microseconds, and a pulse repetition rate of 33kHz. The plates subject to light treatment were irradiated three timesat 0, 72 and 144 hours with a radiant exposure of 7.56 J/cm² during eachirradiation session (i.e., each irradiation session was 42 minutes withan incubation period therebetween of 30 minutes).

After irradiation protocols were completed, the plates were placedupside down in the anaerobic chamber and incubated at 37° C. for 24hours. The plates were photographed, colonies counted and statistics ofpercent survival recorded.

Example 5

In a fifth experiment, the GBS were assigned to six experimental groups:(1) GBS only (control); (2) GBS and light; (3) GBS supplemented with0.02 mg/mL PPIX (incubated); (4) GBS supplemented with 0.02 mg/mL PPIX(incubated) and light; (5) GBS supplemented with 0.02 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.02 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 37.

As can be seen in FIG. 37, irradiation of GBS without the addition ofany PPIX showed a minimal decrease in percent GBS survival when comparedto the control, i.e., approximately 97%, which demonstrates that GBSdoes not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.02 mg/mL PPIX (incubated), but without anyirradiation, did not show much decrease in percent GBS survival whencompared to the control, i.e., approximately 92%. In addition,irradiation of GBS supplemented with 0.02 mg/mL PPIX (incubated) showeda small decrease in percent GBS survival when compared to the control,i.e., approximately 68%. This result is different than those seen inFIGS. 33 and 34 because the bacteria was incubated at 37° C. forapproximately thirty minutes before irradiation, which gives them moretime to grow, hence starting with an overall increased CFU/mL comparedto Examples 1 and 2 where irradiation was commenced immediately afterdilutions were made. This is reflected in the higher percent of remnantcolonies obtained in this experiment. GBS supplemented with 0.02 mg/mLPPIX (incubated and washed) showed some decrease in percent GBS survivalwhen compared to the control, i.e., approximately 92%. It is believedthat there is a slight decrease in percent GBS survival because PPIX mayhave some minimal suppressive effects that may be concentrationdependent. Further, irradiation of GBS supplemented with 0.02 mg/mL PPIX(incubated and washed) showed a significant decrease in percent GBSsurvival when compared to the control, i.e., approximately 18%.

Overall, the decrease in percent GBS survival was greatest for GBSsupplemented with PPIX that was incubated and washed prior toirradiation, which suggests that a sufficient quantity of PPIX wasinternalized into the GBS during the incubation period. It should benoted that the percent GBS survival for GBS supplemented with PPIX thatwas incubated prior to irradiation was greater than the percent GBSsurvival for GBS supplemented with PPIX that was incubated and washedprior to irradiation. This difference is believed to be caused by thefact that incubation with porphyrin has some minimal effect on bacterialsurvival and, thus, it is likely that more dead bacteria will be washedduring the washing step of the incubated sample resulting in a reducedstarting CFU/mL.

Example 6

In a sixth experiment, the GBS were assigned to six experimental groups:(1) GBS only (control); (2) GBS and light; (3) GBS supplemented with0.02 mg/mL PPIX (incubated); (4) GBS supplemented with 0.02 mg/mL PPIX(incubated) and light; (5) GBS supplemented with 0.02 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.02 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 38. Itshould be noted that the results for Example 6 are similar to those ofExample 5, which is indicative of the reproducibility of the data.

Example 7

In a seventh experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.05 mg/mL PPIX (incubated); (4) GBS supplemented with 0.05 mg/mLPPIX (incubated) and light; (5) GBS supplemented with 0.05 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.05 mg/mL PPIX(incubated and washed)) and light. The results are shown in FIG. 39.

As can be seen in FIG. 39, irradiation of GBS without the addition ofany PPIX showed a minimal decrease in percent GBS survival when comparedto the control, i.e., approximately 80%, which demonstrates that GBSdoes not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.05 mg/mL PPIX (incubated), but without anyirradiation, did not show much decrease in percent GBS survival whencompared to the control, i.e., approximately 72%. In addition,irradiation of GBS supplemented with 0.05 mg/mL PPIX (incubated) showeda small decrease in percent GBS survival when compared to the control,i.e., approximately 60%. As explained above, this result is differentthan those seen in FIGS. 33 and 34 because the bacteria was incubated at37° C. for approximately thirty minutes before irradiation, which givesthem more time to grow, hence starting with an overall increased CFU/mLcompared to Examples 1 and 2 where irradiation was commenced immediatelyafter dilutions were made. This is reflected in the higher percent ofremnant colonies obtained in this experiment. GBS supplemented with 0.05mg/mL PPIX (incubated and washed) showed some decrease in percent GBSsurvival when compared to the control, i.e., approximately 92%. Again,it is believed that there is a slight decrease in percent GBS survivalbecause PPIX may have some minimal suppressive effects that may beconcentration dependent. Further, irradiation of GBS supplemented with0.05 mg/mL PPIX (incubated and washed) showed a significant decrease inpercent GBS survival when compared to the control, i.e., approximately18%.

Overall, the decrease in percent GBS survival was greatest for GBSsupplemented with PPIX that was incubated and washed prior toirradiation, which suggests that the PPIX was internalized into the GBSduring the incubation period. It should be noted that the percent GBSsurvival for GBS supplemented with PPIX that was incubated prior toirradiation was greater than the percent GBS survival for GBSsupplemented with PPIX that was incubated and washed prior toirradiation. This difference is believed to be caused by the fact thatincubation with porphyrin has some minimal effect on bacterial survivaland, thus, it is likely that more dead bacteria will be washed duringthe washing step of the incubated sample resulting in a reduced startingCFU/mL.

Example 8

In an eighth experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.05 mg/mL PPIX (incubated); (4) GBS supplemented with 0.05 mg/mLPPIX (incubated) and light; (5) GBS supplemented with 0.05 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.05 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 40. Itshould be noted that the results for Example 8 are similar to those ofExample 7, which is indicative of the reproducibility of the data.

Example 9

In a ninth experiment, the GBS were assigned to six experimental groups:(1) GBS only (control); (2) GBS and light; (3) GBS supplemented with0.05 mg/mL PPIX (incubated); (4) GBS supplemented with 0.05 mg/mL PPIX(incubated) and light; (5) GBS supplemented with 0.05 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.05 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 41.

As can be seen in FIG. 41, irradiation of GBS without the addition ofany PPIX showed a minimal decrease in percent GBS survival when comparedto the control, i.e., approximately 94%, which demonstrates that GBSdoes not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.05 mg/mL PPIX (incubated), but without anyirradiation, did not show much decrease in percent GBS survival whencompared to the control, i.e., approximately 90%. In addition,irradiation of GBS supplemented with 0.05 mg/mL PPIX (incubated) showeda large decrease in percent GBS survival when compared to the control,i.e., approximately 20%. GBS supplemented with 0.05 mg/mL PPIX(incubated and washed) also showed some decrease in percent GBS survivalwhen compared to the control, i.e., approximately 54%. Again, it isbelieved that there is a slight decrease in percent GBS survival becausePPIX may have some minimal suppressive effects that may be concentrationdependent. Further, irradiation of GBS supplemented with 0.05 mg/mL PPIX(incubated and washed) showed a significant decrease in percent GBSsurvival when compared to the control, i.e., approximately 6%, whichsuggests that the PPIX was internalized into the GBS during theincubation period.

Example 10

In a tenth experiment, the GBS were assigned to six experimental groups:(1) GBS only (control); (2) GBS and light; (3) GBS supplemented with0.05 mg/mL CP III (incubated); (4) GBS supplemented with 0.05 mg/mL CPIII (incubated) and light; (5) GBS supplemented with 0.05 mg/mL CP III(incubated and washed); and (6) GBS supplemented with 0.05 mg/mL CP III(incubated and washed) and light. Because the CP III was solubilized inethanol, two additional experimental groups were included to testwhether the quantity of ethanol used as a solvent had any significanteffect on bacterial growth suppression: (7) ethanol only and (8) ethanoland light. The results are shown in FIG. 42.

As can be seen in FIG. 42, irradiation of GBS without the addition ofany CP III showed a small decrease in percent GBS survival when comparedto the control, i.e., approximately 82%, which again demonstrates thatGBS does not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.05 mg/mL CP III (incubated), but without anyirradiation, showed a small decrease in percent GBS survival whencompared to the control, i.e., approximately 90%. In addition,irradiation of GBS supplemented with 0.05 mg/mL CP III (incubated)showed complete bacterial kill, i.e., 0% GBS survival. GBS supplementedwith 0.05 mg/mL CP III (incubated and washed) showed minimal decrease inpercent GBS survival when compared to the control, i.e., approximately72%. Further, irradiation of GBS supplemented with 0.05 mg/mL CP III(incubated and washed) showed complete bacterial kill, i.e., 0% GBSsurvival, which suggests that the CP III was internalized into the GBSduring the incubation period. It should also be noted that ethanol didnot have any significant contribution to the observed suppressiveeffect.

Example 11

In an eleventh experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.08 mg/mL PPIX (incubated); (4) GBS supplemented with 0.08 mg/mLPPIX (incubated) and light; (5) GBS supplemented with 0.08 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.08 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 43.

As can be seen in FIG. 43, irradiation of GBS without the addition ofany PPIX showed a minimal decrease in percent GBS survival when comparedto the control, i.e., approximately 98%, which demonstrates that GBSdoes not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.08 mg/mL PPIX (incubated), but without anyirradiation, did not show much decrease in percent GBS survival whencompared to the control, i.e., approximately 84%. In addition,irradiation of GBS supplemented with 0.08 mg/mL PPIX (incubated) showedsome decrease in percent GBS survival when compared to the control,i.e., approximately 52%. GBS supplemented with 0.08 mg/mL PPIX(incubated and washed) showed a small decrease in percent GBS survivalwhen compared to the control, i.e., approximately 76%. Again, it isbelieved that there is a slight decrease in percent GBS survival becausePPIX may have some minimal suppressive effects that may be concentrationdependent. Further, irradiation of GBS supplemented with 0.08 mg/mL PPIX(incubated and washed) showed some decrease in percent GBS survival whencompared to the control, i.e., approximately 40%, which suggests that atleast some of the PPIX was internalized into the GBS during theincubation period.

Example 12

In a twelfth experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.08 mg/mL PPIX (incubated); (4) GBS supplemented with 0.08 mg/mLPPIX (incubated) and light; (5) GBS supplemented with 0.08 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.08 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 44. Itshould be noted that the results for Example 12 are similar to those ofExample 11, which is indicative of the reproducibility of the data.

Example 13

In a thirteenth experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.2 mg/mL PPIX (incubated); (4) GBS supplemented with 0.2 mg/mLPPIX (incubated) and light; (5) GBS supplemented with 0.2 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.2 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 45.

As can be seen in FIG. 45, irradiation of GBS without the addition ofany PPIX showed a minimal decrease in percent GBS survival when comparedto the control, i.e., approximately 95%, which demonstrates that GBSdoes not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.2 mg/mL PPIX (incubated), but without anyirradiation, did not show much decrease in percent GBS survival whencompared to the control, i.e., approximately 80%. In addition,irradiation of GBS supplemented with 0.2 mg/mL PPIX (incubated) showedsome decrease in percent GBS survival when compared to the control,i.e., approximately 50%. GBS supplemented with 0.2 mg/mL PPIX (incubatedand washed) showed a small decrease in percent GBS survival whencompared to the control, i.e., approximately 78%. Again, it is believedthat there is a slight decrease in percent GBS survival because PPIX mayhave some minimal suppressive effects that may be concentrationdependent. Further, irradiation of GBS supplemented with 0.2 mg/mL PPIX(incubated and washed) showed a significant decrease in percent GBSsurvival when compared to the control, i.e., approximately 10%, whichsuggests that the PPIX was internalized into the GBS during theincubation period.

Example 14

In a fourteenth experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.2 mg/mL PPIX (incubated); (4) GBS supplemented with 0.2 mg/mLPPIX (incubated) and light; (5) GBS supplemented with 0.2 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.2 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 46. Itshould be noted that the results for Example 14 are similar to those ofExample 13, which is indicative of the reproducibility of the data.

Example 15

In a fifteenth experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.2 mg/mL PPIX (incubated); (4) GBS supplemented with 0.2 mg/mLPPIX (incubated) and light; (5) GBS supplemented with 0.2 mg/mL PPIX(incubated and washed); and (6) GBS supplemented with 0.2 mg/mL PPIX(incubated and washed) and light. The results are shown in FIG. 47. Itshould be noted that the results for Example 15 are similar to those ofExamples 13 and 14, which is again indicative of the reproducibility ofthe data.

Example 16

In a sixteenth experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.2 mg/mL CP III (incubated); (4) GBS supplemented with 0.2 mg/mLCP III (incubated) and light; (5) GBS supplemented with 0.2 mg/mL CP III(incubated and washed); and (6) GBS supplemented with 0.2 mg/mL CP III(incubated and washed) and light. Because the CP III was solubilized inethanol, two additional experimental groups were included to testwhether the quantity of ethanol used as a solvent had any significanteffect on bacterial growth suppression: (7) ethanol only and (8) ethanoland light. The results are shown in FIG. 48.

As can be seen in FIG. 48, irradiation of GBS without the addition ofany CP III showed a small decrease in percent GBS survival when comparedto the control, i.e., approximately 96%, which again demonstrates thatGBS does not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.2 mg/mL CP III (incubated), but without anyirradiation, showed a small decrease in percent GBS survival whencompared to the control, i.e., approximately 82%. In addition,irradiation of GBS supplemented with 0.2 mg/mL CP III (incubated) showedcomplete bacterial kill, i.e., 0% GBS survival. GBS supplemented with0.2 mg/mL CP III (incubated and washed) showed minimal decrease inpercent GBS survival when compared to the control, i.e., approximately80%. Further, irradiation of GBS supplemented with 0.2 mg/mL CP III(incubated washed) showed complete bacterial kill, i.e., 0% GBSsurvival, which suggests that the CP III was internalized into the GBSduring the incubation period. It should also be noted that ethanol didnot have any significant contribution to the observed suppressiveeffect.

Example 17

In a seventeenth experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.2 mg/mL CP III (incubated); (4) GBS supplemented with 0.2 mg/mLCP III (incubated) and light; (5) GBS supplemented with 0.2 mg/mL CP III(incubated and washed); and (6) GBS supplemented with 0.2 mg/mL CP III(incubated and washed) and light. Because the CP III was solubilized inethanol, two additional experimental groups were included to testwhether the quantity of ethanol used as a solvent had any significanteffect on bacterial growth suppression: (7) ethanol only and (8) ethanoland light. The results are shown in FIG. 49.

As can be seen in FIG. 49, irradiation of GBS without the addition ofany CP III showed a small decrease in percent GBS survival when comparedto the control, i.e., approximately 98%, which again demonstrates thatGBS does not synthesize a sufficient amount of porphyrin. Also, GBSsupplemented with 0.2 mg/mL CP III (incubated), but without anyirradiation, showed a small decrease in percent GBS survival whencompared to the control, i.e., approximately 82%. In addition,irradiation of GBS supplemented with 0.2 mg/mL CP III (incubated) showedcomplete bacterial kill, i.e., 0% GBS survival. GBS supplemented with0.2 mg/mL CP III (incubated and washed) showed a small decrease inpercent GBS survival when compared to the control, i.e., approximately72%. Further, irradiation of GBS supplemented with 0.2 mg/mL CP III(incubated and washed) showed some decrease in percent GBS survival whencompared to the control, i.e., approximately 42%. It should also benoted that ethanol did not have any significant contribution to theobserved suppressive effect.

Example 18

In an eighteenth experiment, the GBS were assigned to six experimentalgroups: (1) GBS only (control); (2) GBS and light; (3) GBS supplementedwith 0.2 mg/mL CP III (incubated); (4) GBS supplemented with 0.2 mg/mLCP III (incubated) and light; (5) GBS supplemented with 0.2 mg/mL CP III(incubated and washed); and (6) GBS supplemented with 0.2 mg/mL CP III(incubated and washed) and light. Because the CP III was solubilized inethanol, two additional experimental groups were included to testwhether the quantity of ethanol used as a solvent had any significanteffect on bacterial growth suppression: (7) ethanol only and (8) ethanoland light. The results are shown in FIG. 50. It should be noted that theresults for Example 18 are similar to those of Example 17, which isindicative of the reproducibility of the data.

V. Electron Microscopic Evidence of Disruption and Damage to CellMembrane of MRSA after Irradiation with Pulsed Blue Light

Further testing was performed using an electron microscope to determinethe impact on the cell membrane of MRSA when irradiated with pulsed bluelight using printed LED flexible lamps.

Methods

MRSA was cultured in Tryptic soy broth (TSB) and divided into two groupsof 1×10⁸ CFU/mL. Each group was separately plated on Tryptic soy agar(TSA). One group was irradiated with a sub-lethal dose of 2.5 J/cm² of33% pulsed 450 nm blue light, while the other group served asnon-irradiated control. A sub-lethal dose was used to reduce theantimicrobial effect of blue light; otherwise, it would havephoto-eradicated the entire bacterial culture. Irradiated andnon-irradiated MRSA cultures were then placed in a 37° C. incubator for24 hours.

MRSA colonies obtained from both groups were resuspended in TSB, fixedand then processed for Transmission Electron Microscopy (TEM) and lightmicroscopy using standard procedures, as shown in FIG. 55. Samples werethen sectioned and studied using a Zeiss light microscope and a TechnaiTEM.

Results

Light microscopy of control and irradiated (i.e., treated) MRSA showedthat the samples were appropriately processed for TEM imaging. FIG. 56are light micrographs showing non-irradiated control MRSA (left panel)and irradiated MRSA (right panel).

TEM of irradiated MRSA clearly revealed structural and morphologicalchanges in the treated group compared to the control group. While thecell walls of non-irradiated control MRSA appeared normal, smooth andstable, irregularities were seen in the cell walls of irradiated MRSA,which appeared disrupted and fractured, with breaks in portions of thecell membranes. FIG. 57 provides evidence showing normal cell divisionin non-irradiated control MRSA (A and C) and disruption of normal celldivision in MRSA irradiated with pulsed 450 nm blue light (B and D). Ofnote is the structural modification of the nuclear material ofirradiated MRSA [Magnification=110,000] (B and D).

Also observed were cytoplasmic evidence of metabolic irregularities inthe irradiated (i.e., treated) group. FIG. 58 provides evidence showingstructural damage to irradiated MRSA (B) compared to non-irradiatedcontrol MRSA (A). FIG. 58 also shows normal cell division innon-irradiated control MRSA (A) compared to irradiated MRSA (B)[Magnification=67,000].

Control cells evinced high levels of metabolic activities in theircytoplasm and the nuclei, and this is evidenced by the fine distributionof intracellular content throughout the cell, as shown in FIG. 57 (A, C)and FIG. 58 (A). Contrary to this observation, the treated cells shownin FIG. 57 (B, D) and FIG. 58 (B) have very dense clumps of nuclearmaterial, suggestive of oxidative stress and low metabolism.Furthermore, the non-irradiated control MRSA of FIG. 57 (A, C) and FIG.58 (A) can be seen undergoing normal cell division as expected of ahealthy rapidly replicating bacterium, while cell division can be seento be irregular and structurally disrupted in the irradiated MRSA ofFIG. 57 (B, D) and FIG. 58 (B). As shown in FIG. 57 (A), early in thecell replication cycle, non-irradiated control MRSA can be seen withindentations of the membrane (arrowed), an indication that the cell isin the early stage of dividing or replicating. Later in the cellreplication cycle, non-irradiated control MRSA were seen with clear-cutcleavage furrows, as shown in FIG. 57 (C) and FIG. 58 (A). Incomparison, the cell membrane of irradiated MRSA appeared disrupted andirregular with no evidence of cell replication, as shown in FIG. 57 (Band D) and FIG. 58 (B).

CONCLUSION

These findings indicate that disruption of cell replication, andstructural damage of bacterial cell walls, are two other mechanismsaccounting for the antimicrobial effect of pulsed blue light on MRSA. Itis believed that these findings would also apply to other types ofmicroorganisms.

VI. Detection of Emission Spectra of Light Absorbing Pigment“Porphyrins” in P. acnes

P. acnes gives off a florescence in the red spectrum of 600-700 nm(typically 620 nm to 640 nm) when illuminated with pulsed purple or bluelight. Without being bound by any one theory, it is believed thatporphyrins are excited (i.e., optically pumped) with each light pulseand, upon return to their ground state, create an oxidation reactionthat produces free radicals which subsequently destroy mitochondrialmembranes, DNA or other cellular structures, as described above. Thus,if a photo sensor is used to detect red or other wavelengths given offby the cells undergoing irradiation, then the feedback can be used tocontrol the dose of light, e.g., irradiation would take place until theporphyrin level had reached a minimum point and would not begin untilporphyrin recovery took place. This may take place as part of thereplication cycle of the microorganism.

Testing was performed to detect the emission spectra of light absorbingpigment porphyrins in P. acnes.

A 72 hour culture of P. acnes was centrifuged at 13,300 rpm for fiveminutes, supernatant discarded and re-suspended in 1 mL saline. It wasthen washed again by centrifuging at 13,300 rpm for 5 minutes with 1 mLsaline (washing ensures that no other factors—aside from the innateporphyrins found in the P. acnes cells—would contribute to thedetection). The optical density was adjusted using McFarland standard to0.8 to 1.0 at 625 nm for a concentration of 108 CFU/mL. A volume of 10μl of this solution was placed on concanavalin A coated dishes andexcited as described below.

As illustrated in FIG. 23, the set-up used for irradiating the bacteriaemployed a 458 nm Ar-Ion laser assembly (Edmund optics, USA) deliveringcontinuous wave (CW) light with spectral bandwidth of less than 1 nm anda total power of 150 mW. The original, divergent laser beam wascollimated using a lens (focal length 35 mm) placed about 22 mm from thelaser aperture. The final beam diameter was 15 mm with a cross-sectionalarea of 3 cm², which approximately matched the area of the dish coatedwith concanavalin A. The sample was placed on a translation stagepositioned about 25 cm from the collimating lens.

The sample was placed onto an inverted microscope (Axio Observer—Z1,Carl Zeiss, Oberkochen, Germany) and illuminated through the back portof the microscope using a continuous wave Ar-Ion laser (Edmund Optics)with a 458 nm wavelength. The excitation light (0.5 mW) was focused by a63× water immersion objective with numerical aperture (NA) of 1.2. Themicroscope scanning head consisted of a computer-controlled OptiMiSTruLine (Aurora Spectral Technologies, Milwaukee, Wis., USA). Thedetection was done using a modified OptiMiS detection module (acommercial prototype of OptiMiS d-Lux), which included a galvanometricscanner used to descan the fluorescence beam which then falls on anelectron multiplying charged coupled device (iXon3, Andor, Belfast, UK).This descanning concept allowed the use of OptiMiS as a confocal (ratherthan a two-photon) microscope. This in turn reduced the laser-inducedphoto-bleaching of the cellular fluorescence, which was critical inthese experiments given the low intensity of the signal detected. Inorder to capture full spectrum images, the sample was scanned line byline. The line images were further reconstructed resulting in an imagefor each wavelength channel (total of 300 channels).

The extraction of the porphyrin spectrum was performed by firstmeasuring the spectrum of the emission from the bacteria and thespectrum of the media outside of the bacteria. Next, the spectrum of themedia was subtracted from the bacterial emission spectrum whileconsidering the emission ratios between the bacteria and the outsidemedia. Finally, the resulting spectrum was fitted with two Gaussians asthe emission has two humps.

FIG. 24A depicts the porphyrin emission spectrum extracted from P. acnesexcited with 458 nm light and detected using OptiMiS TruLine (AuroraSpectral Technologies, Milwaukee, Wis.). The peak wavelengths of theresulting emission were 619 nm and 670 nm for the two apparent humps.This emission is characteristic of cytochrome C which most likely ispresent in the acne bacteria. However, it may still contain someemission from different porphyrins such as coproporphyrins which mayparticipate in the bacterial eradication mechanism. It is proposed thatthe free radical production by peroxides generated from porphyrinsexposed to purple or blue light may alter DNA structure and lead todestruction of the bacteria during its replicative phase. Thus, thetiming of light exposures during the replicative phase may havesignificant impact on the kill rates.

FIG. 24B demonstrates the fluorescence spectra of P. acnes at varioustimes during irradiation from zero to 30 minutes (measured at T=1, 10,20 and 30 minutes). It can be readily seen that the fluorescencegradually depletes over time and thus can act as an indicator ofporphyrin depletion at the cellular level. The porphyrin depletionfollows the dose parameters (approximately 30 minutes of irradiation at3 mW/cm²) which were found to be optimal for photoeradication ofbacteria. These experiments were performed in vitro, in a petri dish inagar, illuminated with 450 nm printed LEDs on a flexible substrate. Theoutput was pulsed at a duty factor of 33% (10 microseconds on and 20microseconds off) at an irradiance of 3 mW/cm². A calibrated oceanoptics OCEAN-FX-VIS-NIR-ES spectrometer was used to record thefluorescence. The dish was held in a light tight housing. The blue 450nm excitation light was filtered to eliminate all wavelengths beyond 420and 480 nm. A filter was used below the dish to eliminate anywavelengths below 510 nm. The extraction of the porphyrin spectrum wasperformed by first measuring the spectrum of the emission from thebacteria and the spectrum of the media outside of the bacteria. Next,the spectrum of the media was subtracted from the bacterial emissionspectrum while considering the emission ratios between the bacteria andthe outside media. The spectra were then plotted.

FIG. 24C depicts the porphyrin emission spectrum extracted from a sampleof Protoporphyrin IX excited with 450 nm printed LEDs on PET substrateand detected using an ocean optics OCEAN-FX-VIS-NIR-ES spectrometer inthe same test set-up as FIG. 24B. The peak wavelengths of the resultingemission were 620 nm and 670 nm and 750 nm for the three apparent humps.This emission is similar to the initial porphyrin emission spectrumextracted from P. acnes excited with 458 nm light (FIG. 24A); however,it has a broader emission in the near infrared range.

Dosage can thus be optimized by detecting the emission spectrum ofporphyrins or other emissions from the targeted bacteria, measuringfluorescence depletion or recovery, determining the optimal excitationperiods and recovery between treatment sessions, and calculating idealirradiation parameters, including dosage, irradiance, fluence andtreatment schedule. It is also possible that killing of differentstrains of bacteria can be optimized using this method. Idealwavelengths to measure porphyrin depletion are shown to be in the620-640 nm range and the 670 nm range from the data presented, whichcorrelates well with our data shown in FIGS. 24A, 24B and 24C. Idealwavelengths to measure other types of emissions can be determined by oneskilled in the art.

VII. Exemplary Light Sources

Various types of light sources may be used to provide light energy inaccordance with the present invention. For example, the light source maycomprise various types of lasers, LEDs, PLEDs/LEPs), QDLEDs, orfluorescent tubes emitting light in the purple or blue spectral region(and optionally the red or infrared spectral region). The light sourcemay directly illuminate the skin, tissue or other surface to beirradiated, such as a contaminated environment or food to be exposed topulsed light. The light source may also be coupled by fiber optics ordirectly mounted to the edge of a lens-diffuser that redirects the lightto the surface to be irradiated. These diffusers may contain numerousoptical elements designed to diffuse and distribute the light evenlyacross the surface to be irradiated. These may include Fresnel lenses,various light pipes, irregularities and additional lens-like structuresdesigned to pipe the light source and distribute it evenly over thesurface of the lens-diffuser assembly. In order to improve theextraction or out-coupling of light, the device may include an internalscattering layer of high index particles such as TiOx in a transparentphotoresist or a micro-lens array (MLA) layer. The light sources mayalso be placed directly behind the diffuser or Fresnel lens at asufficient distance to allow the production of a substantially uniformbeam to be applied to the surface to be irradiated. The light source maybe a combination of white light and purple or blue light, whereupon thepurple or blue light is pulsed and the white light is continuous wave(CW). These different wavelengths can be applied simultaneously orsequentially to irradiate an infected environment or food.

A preferred light source is provided in the form of a very thin layeredstructure. An example of this type of light source is shown generally asreference numeral 10 in FIG. 25. Light source 10 is comprised of aplurality of layers 12 including a flexible light emitter 40 locatedbetween an anode 50 and a cathode 60. A suitable power source isconnected to light source 10. Preferably, direct current (DC) or pulsedDC is used to power light source 10. Light source 10 is substantiallyplanar in its form, although it is preferably flexible and is morepreferably conformable such that it may conform to the contours of thebody, surfaces within a contaminated environment, or surfaces inside ofa refrigeration or food display system. The overall thickness of lightsource 10 is typically about 10 mm or less (e.g., about 10, 9, 8, 7, 6,5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 mm or less).

Light source 10 produces light with an intensity that is substantiallyconstant across the surface of the device so as to provide substantiallyuniform light emission. As described in more detail below, the lightsource may comprise, for example, organic light emitting diodes (OLEDs)or printed light emitting diodes (printed LEDs) (organic or inorganic)commonly referred to as LED ink. It can be appreciated that the lightsource is capable of decreasing hot spots on the surface of a subject'sskin or tissue to provide a safer delivery of light to a subject. Also,a substantially uniform dose of light across the surface of the deviceensures that all of the skin, tissue, surface or food is effectivelytreated with the same dose of light.

FIG. 22 shows an example of a light source that includes a plurality oflight emitting diodes 42 printed on a flexible PET (polyethyleneterephthalate)—ITO coated film 44 (referred to herein as a printed LEDflexible lamp or a lighted substrate). In this example, the lightemitting diodes 42 are printed in hexagon-shaped LED clusters at adensity of 2.5 LEDS/mm². Of course, in other examples, the LED clusterscould have other shapes, the LEDS could be printed across the entiresurface of the film, and/or the LEDs could be printed in other densitiesin accordance with the present invention.

In one embodiment, flexible light emitter 40 comprises a printed LEDfilm that is laminated to a quantum dot film. In this embodiment, all ofthe light emitting diodes of the printed LED film (such as lightemitting diodes 42 shown in FIG. 22) are configured to emit purple orblue light. The quantum dot film comprises a slurry of quantum dots(including cadmium-free quantum dots (CFQDs)) coated on a flexible filmand then another flexible film is placed thereon in order to seal thequantum dots between the two films. Alternatively, the CFQDs could becoated on the printed LED substrate with a barrier coating or filmapplied to the outer side in order to prevent oxidation of the CFQDs.The quantum dots are configured to convert purple or blue light to redor infrared light upon receipt of the pulsed purple or blue light fromthe printed LED film so as to maintain the same pulse characteristicsfor both the red or infrared light and the purple or blue light. Variousdensities of quantum dots can be used to control the percentage of lightconverted from purple or blue light to red or infrared light. In oneembodiment, the percentages of purple or blue and red or infrared lightrange from 20% purple or blue light/80% red or infrared light to 80%purple or blue light/20% red or infrared light. Of course, otherpercentages of purple or blue and red or infrared light could also beused as desired for a certain application. Preferably, the purple orblue light emitting diodes and the quantum dots are evenly distributedacross the surface of the device so as to provide a substantiallyuniform emission of purple or blue and red or infrared light.

In another embodiment, flexible light emitter 40 comprises only aprinted LED film (i.e., a quantum dot film is not used in thisembodiment). In this embodiment, the light emitting diodes of theprinted LED film (such as light emitting diodes 42 shown in FIG. 22)comprise a combination of purple or blue and red or infrared lightemitting diodes that are printed on a flexible film (such as flexiblefilm 44 shown in FIG. 22) in a checkerboard or grid-like pattern. Therespective number of purple or blue and red or infrared light emittingdiodes can be chosen to achieve the desired levels of purple or blue andred or infrared light. In one embodiment, the percentages of purple orblue and red or infrared light preferably range from 20% purple or bluelight/80% red or infrared light to 80% purple or blue light/20% red orinfrared light. Of course, other percentages of purple or blue and redor infrared light could also be used as desired for a certainapplication. Preferably, the purple or blue and red or infrared lightemitting diodes are evenly distributed across the surface of the deviceso as to provide a substantially uniform emission of purple or blue andred or infrared light.

It should be understood that the irradiance and radiant exposure of eachof the purple or blue light and red or infrared light will be apercentage of the total irradiance and radiant exposure discussed above,as determined by the density of the quantum dots (for the firstembodiment discussed above) or the respective numbers of purple or blueand red or infrared light emitting diodes (for the second embodimentdiscussed above), or the drive current applied respectively to thepurple or blue and red or infrared light emitting diodes by the drivingdevice. As such, the desired levels of purple or blue light and red orinfrared light may be selectively achieved for a particular lighttreatment.

In another embodiment, flexible light emitter 40 comprises OLEDslaminated to a quantum dot film, wherein the OLEDs emit purple or bluelight and the quantum dot film converts a portion of the purple or bluelight to red or infrared light. In yet another embodiment, flexiblelight emitter 40 comprises OLEDS that emit both purple or blue and redor infrared light. It can be appreciated that these embodiments aresimilar to the two embodiments described above, with the exception thatthe printed LED film is replaced with the OLEDs. In yet otherembodiments, flexible light emitter 40 comprises printed LED film orOLEDS that emit only purple or blue light.

The light source may be in the form of an array, patch, pad, mask, wrap,fiber, bandage or cylinder, for example. The light source may have avariety of shapes and sizes. For example, the light source may besquare, rectangular, circular, butterfly-shaped, elliptical,clover-shaped, oblong, crescent/moon-shaped, or any other shape that issuitable for a particular application. Examples of suitable lightsources are generally illustrated in FIGS. 26A and 26B, as well as thelight devices shown in FIGS. 51 and 52. The overall surface area of oneside of the light source may range from, for example, 1 cm² to 1 m², orin panels placed together to cover larger areas, although typically thesurface area is about 1 to 2000 cm² (e.g., about 1, 4, 9, 16, 25, 36,49, 64, 81, 100, 121, 144, 169, 196, 225, 289, 324, 361, 400, 441, 484,529, 576, 625, 676, 729, 784, 841, 900, 961, 1024, 1089, 1156, 122,1296, 1369, 1444, 1521, 1600, 1681, 1764, 1849, 1936 or 2000 cm² or somerange therebetween). The light source is thus well adapted to be appliedto various areas of the subject's body, for example, the face, forehead,cheek, back, wrist and the like. The light source may also be appliedother types of surfaces, such as surfaces within a contaminatedenvironment or surfaces inside of a refrigeration or food displaysystem.

The various elements/layers of the light source will now be described ingreater detail.

Substrate

In some embodiments, the light source includes a substrate. Thesubstrate may be any substance capable of supporting the various layersof the light source. The substrate is preferably flexible and/orconformable to a surface in which the light source will be used, such asthe contours of a subject's body, surfaces within a contaminatedenvironment, or surfaces inside of a refrigeration, food display orprocessing system. The substrate can comprise, for example, an inorganicmaterial, an organic material, or a combination of inorganic and organicmaterials. The substrate may be, for example, made from metals, plasticsor glass. The substrate may be any shape to support the other componentsof the light source, for example, the substrate may be substantiallyflat or planar, curved, or have portions that are substantially flatportions and curved portions. Most preferably, the substrate istransparent, flexible, and conformable in nature. Ideally, the materialis a latex-free, non-toxic, non-allergenic material, which is resistantto UV, sunlight and most infection control products.

As used herein, the term “transparent” generally means transparency forlight and includes both clear transparency as well as translucency.Generally, a material is considered transparent if at least about 50%,preferably about 60%, more preferably about 70%, more preferably about80% and still more preferably about 90% of the light illuminating thematerial can pass through the material. In contrast, the term “opaque”generally refers to a material in which the light is substantiallyabsorbed or reflected, e.g., at least 90% of the light is absorbed orreflected, and typically at least 95% of the light is absorbed orreflected.

In some embodiments, the substrate may be comprised of a silicon-basedmaterial, rubber, thermoplastic elastomers (TTP), or other polymericmaterial, such as polyester, polyethylene terephthalate (PET),polyethylene naphthalate (PEN), polycarbonate, polystyrene, polyacryl,polyether sulfone (PES), etc. Transparent substrates may include, forexample, polyethylene, ethylene-vinyl acetate copolymers, polyimide(PI), polyetherimide (PEI), ethylene-vinyl alcohol copolymers,polypropylene, polystyrene, polymethyl methacrylate, PVC, polyvinylalcohol, polyvinylbutyral, polyether ether ketone, polysulfone,polyether sulfone, as well as fluoropolymers, such as, fluorinatedethylene-propylene (FEP), tetrafluoroethylene-perfluoroalkyl vinyl ethercopolymers, polyvinyl fluoride, tetrafluoroethylene-ethylene copolymers,tetrafluoroethylene-hexafluoropropylene copolymers,polychlorotrifluoroethylene, polyvinylidene fluoride, polyester,polycarbonate, polyurethanes, polyimide or polyether imide.

In another embodiment, the transparent substrate is a polyester film,such as Mylar. In another aspect, the substrate comprises apolyetheretherketone film commercially available from Victrex under thename APTIV. In still another aspect, the substrate is a thin film soldunder the name Flexent by Konica Minolta or flexible glass such asWillow Glass by Dow Corning. Ideally, substrates in direct or indirectcontact with organic layers will have exceptional barrier capabilitiesthat withstand heat, offer flexibility, have sustained reliability andcan be mass produced,

Conductive Layers (Electrodes)

The light source comprises a plurality of conductive layers (i.e.,electrodes), namely, a cathode and an anode. The anode may comprise, forexample, a transparent conductive oxide (TCO), such as, but not limitedto, indium tin oxide (ITO), zinc oxide (Zn0), and the like. The cathodemay also comprise, for example, a thin metal film or fibers such asaluminum, copper, gold, molybdenum, iridium, magnesium, silver, lithiumfluoride and alloys thereof, or a non-metal conductive layer.

Because the light source must emit light through one or both electrodes,at least one of the electrodes must be transparent. The transparentelectrode is positioned on the side of the light source designed to befacing the surface to be irradiated. For a light source intended to emitlight only through the bottom electrode (i.e., the surface-facingelectrode), the top electrode (i.e., the electrode facing away from thesurface) does not need to be transparent. The top electrode may thuscomprise an opaque or light-reflective metal layer having a highelectrical conductivity. Where a top electrode does not need to betransparent, using a thicker layer may provide better conductivity, andusing a reflective electrode may increase the amount of light emittedthrough the transparent electrode by reflecting light back towards thetransparent electrode. Fully transparent light sources may also befabricated, where both electrodes are transparent.

The thickness of each electrode is typically about 200 nm or less (e.g.,about 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30 nm orless). Preferably, the thickness of each electrode is less than 10 nm(e.g., about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.8, 0.6, 0.4, 0.2 nm orless or some range therebetween).

The electrodes are preferably flexible in nature. In some embodiments,the conductive materials of one or both of the electrodes may include,but are not limited to, transparent conductive polymer materials, suchas indiu oxide (ITO), fluorine-doped tin oxide (FTO), ZnO—Ga2O3.ZnO—Al2O3, SnO2—Sb2O3, and polythiophene. In addition, the electrodesmay be comprised of silver or copper grids or bushbars plated on atransparent substrate or silver nanowires or nanoparticles deposited ona substrate with a poly(3,4-ethylenedioxythiophene)poly(styrenesulfonate) (PEDOT:PSS) coating. Additional conductivepolymer layers may be added to improve conductivity.

In one aspect, the transparent conductive electrode may be carbon-based,for example, carbon nanotubes, carbon nanowires, or graphene, and thelike. One preferred electrode (typically for infrared) comprisesgraphene. While one or two layers of graphene are preferred, theelectrode may comprise about 1 to 20 layers of graphene (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 layers orsome range therebetween). The graphene electrode(s) also have the effectof protecting the photoactive layer sandwiched between them fromoxidation. Therefore, environmental stability of the light source can beimproved. The graphene electrode may optionally have a plurality ofplasmonic nanostructures, which may have various morphologies(spherical, rods, discs, prisms, etc.). Exemplary nanostructures includethose made of gold, silver, copper, nickel, and other transition metals,for example gold nanoparticles, silver nanoparticles, coppernanoparticles, nickel nanoparticles, and other transition metalnanoparticles. In general, any electrically conductive materials, suchas oxides and nitrides, of surface plasmonic resonance frequencies inthe visible spectrum can be made into plasmonic nanostructures for thesame purpose. In some embodiments, the plasmonic particles have the sizeof about 1 nm to about 300 nm (e.g., about 10, 50, 100, 150, 200, 250,300 nm, or some range therebetween).

Light Emitter Layer

The light source includes a thin light emitter that may comprise, forexample, OLEDs or printed LEDs (organic or inorganic). The thickness ofthe light emitter is preferably about 2 mm or less (e.g., about 2, 1.8,1.6, 1.4, 1.2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09,0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01 mm or less). Mostpreferably, the flexible light emitter is about 10 to 200 nm inthickness (e.g., about 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50,40, 30, 20, 10 nm, or some range therebetween). The light emitter ispreferably flexible and emits light in response to an electric currentapplied to the anode and cathode.

OLEDs

In some embodiments, the light source may comprise OLEDs in which theflexible light emitter is a thin organic film. As used herein, the term“organic” with respect to OLEDs encompasses polymeric materials as wellas small molecule organic materials that may be used to fabricateorganic opto-electronic devices. Such materials are well known in theart. “Small molecule” refers to any organic material that is not apolymer, and it will be appreciated that “small molecules” may actuallybe quite large. “Small molecules” may include repeat units in somecircumstances. For example, using a long chain alkyl group as asubstituent does not remove a molecule from the “small molecule” class.“Small molecules” may also be incorporated into polymers, for example asa pendent group on a polymer backbone or as a part of the backbone.“Small molecules” may also serve as the core moiety of a dendrimer,which consists of a series of chemical shells built on the core moiety.The core moiety of a dendrimer may be a fluorescent or phosphorescentsmall molecule emitter. A dendrimer may be a “small molecule,” and it isbelieved that all dendrimers currently used in the field of OLEDs aresmall molecules. In general, a “small molecule” has a well-definedchemical formula with a single molecular weight, whereas a polymer has achemical formula and a molecular Tight that may vary from molecule tomolecule.

Generally speaking, in the flexible light emitter, electrons and holesrecombine to radiate photons. The radiative photon energy emitted fromthe flexible light emitter corresponds to the energy difference betweenthe lowest unoccupied molecular orbital (LUMO) level and the highestoccupied molecular orbital (HOMO) level of the organic material. Photonsof lower energy/longer wavelength may be generated by higher-energyphotons through fluorescent or phosphorescent processes.

As described below, the flexible light emitter may optionally includeone or more of a hole injection material (HIM), a hole transportmaterial (HTM), a hole blocking material (HBM), an electron injectionmaterial (EIM), an electron transport material (ETM), an electronblocking material (EBM), and/or an exciton blocking material (ExBM).

In one aspect, the emissive electroluminescent layer may include a holeinjection material (HIM). A HIM refers to a material or unit capable offacilitating holes (i.e., positive charges) injected from an anode intoan organic layer. Typically, a HIM has a HOMO level comparable to orhigher than the work function of the anode, i.e., −5.3 eV or higher.

In another aspect, the emissive electroluminescent layer may include ahole transport material (HTM). A HTM has the characteristic of amaterial or unit capable of transporting holes (i.e., positive charges)injected from a hole injecting material or an anode. A HTM has usuallyhigh HOMO, typically higher than −5.4 eV. In many cases, HIM can alsofunction as HTM, depending on the adjacent layer.

In another aspect, the emissive electroluminescent layer may include ahole blocking material (HBM). A HBM generally refers to a material that,if deposited adjacent to an emitting layer or a hole transporting layerin a multilayer structure, prevents the holes from flowing through.Usually it has a lower HOMO as compared to the HOMO level of the HTM inthe adjacent layer. Hole-blocking layers are frequently inserted betweenthe light-emitting layer and the electron-transport layer.

In another aspect, the emissive electroluminescent layer may include anelectron injection material (EIM). An EIM generally refers to a materialcapable of facilitating electrons (i.e., negative charges) injected froma cathode into an organic layer. The EIM usually has a LUMO levelcomparable to or lower than the working function of the cathode.Typically, the EIM has a LUMO lower than −2.6 eV.

In another aspect, the emissive electroluminescent layer may include anelectron transport material (ETM). An ETM generally refers to a materialcapable of transporting electrons (i.e., negative charges) injected froman EIM or a cathode. The ETM has usually a low LUMO, typically lowerthan −2.7 eV. In many cases, an EIM can serve as an ETM as well,depending on the adjacent layer.

In another aspect, the emissive electroluminescent layer may include anelectron blocking material (EBM). An EBM generally refers to a materialwhich, if deposited adjacent to an emissive or electron transportinglayer in a multilayer structure, prevents the electron from flowingthrough. Usually it has a higher LUMO as compared to the LUMO of the ETMin the adjacent layer.

In another aspect, the emissive electroluminescent layer may include anexciton blocking material (ExBM). An ExBM generally refers to a materialwhich, if deposited adjacent to an emitting layer in a multilayerstructure, prevents the excitons from diffusing through. ExBM shouldhave either a higher triplet level or singlet level as compared to theemitting layer or other adjacent layer.

Exemplary OLED materials are described in Hammond et al., U.S. PublishedPatent Application No. 2010/0179469; Pan et al., U.S. Published PatentApplication No. 2013/0006119; Buchholz et al., PCT Published PatentApplication No. WO 2012/010238; and Adamovich et al., U.S. PublishedPatent Application No. 2007/0247061.

Referring to FIG. 27, a typical sequence of materials found in theflexible light emitter between the anode and the cathode of the OLED isHIM, HTM, emission layer, HBM, and ETM. Another typical sequence ofmaterials is HTM, emission layer, and ETM. Of course, other sequences ofmaterials are also possible. Further, the OLED may comprise one or moreinterlayers.

In one aspect, the flexible light emitter comprises a single layer. Theflexible light emitter may comprise, for example, a conjugated polymerwhich is luminescent, a hole-transporting polymer doped with electrontransport molecules and a luminescent material, or an inert polymerdoped with hole transporting molecules and a luminescent material. Theflexible light emitter may also comprise an amorphous film ofluminescent small organic molecules which can be doped with otherluminescent molecules.

In another aspect, the flexible light emitter may comprise one or moredifferent emissive materials in either the same emission layer or indifferent emission layers. For example, the flexible light emitter maycomprise 5, 4, 3, 2, or 1 radiation emitting materials. The variousdifferent emissive materials may be selected from the emissive materialsdescribed in the references set forth above, but any other suitableemissive material can be employed. If two emissive materials are used inone emission layer, the absorption spectrum of one of the two emissivematerials preferably overlaps with the emission spectrum of the otheremissive material.

The emissive materials may be arranged in stacked layers or side-by-sideconfigurations. The emissive layer may comprise a continuous regionforming a single emitter or a plurality of light emitters. The pluralityof light emitters may emit light with substantially differentwavelengths. The plurality of light emitters may be vertically stackedwithin the emissive layer or they may form a mixture. In someembodiments, a dopant is dispersed within an organic host matrix. In oneembodiment, a layer of quantum dots is sandwiched between two organicthin films.

In another aspect, the flexible light emitter may comprise a pluralityof layers sharing a common anode and/or cathode. In this case,individual layers are stacked one on top of another. The stackedconfiguration may generally include intermediate electrodes disposedbetween adjacent layers such that successive layers share anintermediate electrode, i.e., a top electrode of one layer is the bottomelectrode of another in the stack. The stacked layers may be formed ofdifferent materials, and therefore, different emissions spectra.

The flexible light emitter may be substantially transparent. When mostlytransparent layers are used, a plurality of emissive layers may bevertically stacked without substantially blocking light emission fromindividual layers. The flexible light emitter may comprise a single ormultiple layers, for example, a combination of p- and n-type materials.The p- and n-type materials may be bonded to each other in the layer.The bonding may be ionic or covalent bonding, for example. The multiplelayers of the flexible light emitter may form heterostructurestherebetween.

Printed LEDs

In some embodiments, the light source may comprise printed LEDs (organicor inorganic), i.e., LED ink. With LED ink, each light source is verysmall which enables the LEDs to be positioned in very close proximity toeach other. During fabrication, the LEDs may be printed in a uniformmanner whereby each LED operates as a point source in which the beamsfrom the individual LEDs are substantially parallel to each other toprovide substantially uniform light across the surface of the device.Unlike conventional LEDs, printed LEDs do not need to be positioned asufficient distance from the surface to be irradiated in order todeliver a substantially uniform dose of light. There are several knownmethods for printing such LEDs, as described below.

In one method, a plurality of individual LEDs are suspended anddispersed in a liquid or gel comprising one or more solvents and aviscosity modifier so as to form a diode ink that is capable of beingprinted on a flexible substrate (e.g., through screen printing,flexographic printing and the like). In one aspect, the average surfacearea concentration of LEDs is from about 25 to 50,000 LEDs per squarecentimeter. In general, each LED includes a light emitting region, afirst metal terminal located on a first side of the light emittingregion, and a second metal terminal located on a second side of thelight emitting region. The first and second metal terminals of each LEDmay be electrically coupled to conductive layers (i.e., electrodes) toenable the light emitting region to emit light when energized.

An exemplary light source is shown generally in FIG. 28, wherein onlyfive LEDs are provided in order to simplify the description. As can beseen, this device includes a plurality of conductors 80 a-80 e depositedon a flexible substrate 82. A plurality of LEDs 84 a-84 e are depositedon the conductors 80 a-80 e such that the first metal terminals of theLEDs 84 a-84 e are electrically coupled to the conductors 80 a-80 e. Oneskilled in the art will appreciate that the LEDs 84 a-84 e may be formedof various shapes. Preferably, the LEDs 84 a-84 e settle into a positionover conductors 80 a-80 e such that they maintain their polarity basedon the shape of the LEDs. Next, a plurality of dielectric layers 86 a-86e are deposited over the LEDs 84 a-84 e and the conductors 80 a-80 e, asshown. Another conductor 88 is then deposited over the LEDs 84 a-84 eand dielectric layers 86 a-86 e such that the second metal terminals ofthe LEDs 84 a-84 e are coupled to the conductor 88. One skilled in theart will appreciate that the substrate 82 and conductors 80 a-80 e maybe transparent so that light is emitted from the bottom of the deviceand/or conductor 82 may be transparent so that light is emitted from thetop of the device. Various configurations of printed LEDs that may bemanufactured in accordance with the above method are described inLowenthal et al., U.S. Pat. No. 8,415,879.

In another method, the light source comprises LEDs that are createdthrough a printing process. In this method, a substrate is provided thatincludes a plurality of spaced-apart channels. A plurality of firstconductors are formed on the substrate such that each first conductor ispositioned in one of the channels. Next, a plurality of substantiallyspherical substrate particles are coupled to the first conductors and,then the substantially spherical substrate particles are converted intoa plurality of substantially spherical diodes. The substantiallyspherical diodes may comprise, for example, semiconductor LEDs, organicLEDs encapsulated organic LEDs, or polymer LEDs. A plurality of secondconductors are then formed on the substantially spherical diodes.Finally, a plurality of substantially spherical lenses suspended in apolymer (wherein the lenses and suspending polymer have differentindices of refraction) are deposited over the substantially sphericaldiodes and the second conductors. Thus, in this method, the LED's arebuilt up on the substrate as opposed to being mounted on the substrate.Various configurations of printable LEDs that may be manufactured inaccordance with the above method are described in Ray et al., U.S. Pat.No. 8,384,630.

In an exemplary embodiment, the light source is comprised of printedLEDs having a thickness of 12 μm, silver electrodes each of which has athickness of 5-10 μm with a transparent silver fiber having a thicknessof 0.05-5 μm, and a PET substrate having a thickness of 125 μm.

Micro-Lens Array

The light source may optionally include a light dispersion layer, suchas a micro-lens array. It has been found that one of the key factorsthat limits the efficiency of OLED devices is the inefficiency inextracting the photons generated by the electron-hole recombination outof the OLED devices. Due to the high optical indices of the organicmaterials used, most of the photons generated by the recombinationprocess are actually trapped in the devices due to total internalreflection. These trapped photons never leave the OLED devices and makeno contribution to the light output from these devices. In order toimprove the extraction or out-coupling of light from OLEDs, the devicemay include an internal scattering layer of high index particles such asTiOx in a transparent photoresist or a micro-lens array (MLA) layer.Exemplary MLAs and methods for forming the same are described in Gardneret al., U.S. Published Patent Application No. 2004/01217702; Chari etal. U.S. Pat. No. 7,777,416; Xu et al., U.S. Pat. No. 8,373,341; Yamaeet al., High-Efficiency White OLEDs with Built-up Outcoupling Substrate,SID Symposium Digest of Technical Papers, 43 694 (2012); and Komoda etal., High Efficiency Light OLEDS for Lighting, J. Photopolymer Scienceand Technology, Vol. 25, No. 3 321-326 (2012).

Barrier Layer

The light source may optionally include one or more encapsulation orbarrier layers that isolate the light emitter (or other layers) from anambient environment. The encapsulation or barrier layer is preferablysubstantially impermeable to moisture and oxygen. In general, themoisture and oxygen sensitive components should be enclosed by materialshaving gas permeation properties. The barrier preferably achieves lowwater vapor permeation rates of 10⁻⁴ g/ m²/day or less, 10⁻⁵ g/m²/day orless, and even more preferably about 10⁻⁶ g/m²/day or less.

The encapsulation or barrier layer may be glass or a plastic, forexample. Exemplary materials include a polyetheretherketone filmcommercially available from Victrex under the name APTIV. In stillanother aspect, the substrate is a thin film sold under the name Flexentby Konica Minolta or flexible glass such as Willow Glass by Dow Corning.Ideally, substrates in direct contact with organic layers will haveexceptional barrier capabilities that withstand heat, offer flexibility,have sustained reliability and can be mass produced.

The light source may be further covered with a transparent orsemi-transparent covering. The covering may provide comfort for asubject using the light source. The covering may provide protection tothe light source, keeping dirt and fluid off of the device and providinga cushion to protect the device from impact.

Quantum Dot Layer

In some embodiments, a layer of quantum dots is used between the lightemitting surface and the surface to be irradiated in order to convertall or a portion of the light emission into a different wavelength. Thewavelength is typically down converted to a longer wavelength (Stokesconversion). For example, purple or blue light may be converted into redlight at 630 nm. Quantum dots may contain cadmium or may be organic andcadmium free. Wavelength conversion may occur with many wavelengths thusproviding an additional or multiple wavelengths from a fixed wavelengthsource. Quantum dots may also be embedded into the hydrogel used toconnect the light source to the surface to be irradiated, such as a skinor tissue surface. This may consist of a film or encapsulated quantumdots layered on the surface of the hydrogel or the light emittingsurface of the lamp. Various densities of quantum dots can be used tocontrol the percentage of light converted from one wavelength toanother. For example, testing was performed using 50% and 75% blue lightand 50% and 25% red light in bacterial kill experiments. Thiscombination had an effect on the killing of P. acnes and may be added toenhance the anti-inflammatory effects of the light due to the additionof red light. The quantum dot converts light as it receives the incidentlight so the continuous or pulse characteristic of the light ismaintained.

Bottom and Top Light Emitting Configurations

In some embodiments, the light source has a “bottom” light emittingconfiguration or a “top” light emitting configuration.

FIG. 29 illustrates an exemplary embodiment of a light source 110 with a“bottom” light emitting configuration. Light source 110 comprises aflexible light emitter 140 located between an anode 150 and a cathode160, all of which are formed on a transparent substrate 120. A powersource (not shown) is provided so that DC or pulsed DC is used to powerthe light source. Further, a transparent barrier layer 180 protects theflexible light emitter 140 from moisture and oxygen.

In this embodiment, the flexible light emitter 140 comprises an OLED orprinted LEDs. Both the substrate 120 and the anode 150 are transparent.The substrate is comprised of a transparent silicon rubber. The anode150 is comprised of ITO. Light generated from the flexible light emitter140 is emitted through the transparent anode 150 and substrate 120 suchthat the device has a “bottom” light emitting configuration. The cathode160 is comprised of a conductive metal such as silver. The barrier layeris comprised of Flexent film (Konica Minolta).

FIG. 30 illustrates an exemplary embodiment of a light source 210 with a“top” light emitting configuration. Light source 210 comprises aflexible light emitter 240 located between an anode 250 and a cathode260, all of which are formed on a bottom surface 222 of a substrate 220(i.e., the surface facing towards a surface to be irradiated). A powersource (not shown) is provided so that DC or pulsed DC is used to powerthe light source. Further, a transparent barrier layer 280 protects theflexible light emitter 240 from moisture and oxygen.

In this embodiment, the flexible light emitter 240 comprises an OLED orprinted LEDs. The substrate 220 comprises a Mylar film and a silvernanolayer is coated on its bottom side to form the cathode 260 of thelight source. The silver nanolayer is highly reflective to the lightgenerated by the flexible light emitter 240 such that the light isdirected towards the surface to be irradiated. Both the barrier layer280 and the anode 250 are transparent. The barrier layer 280 iscomprised of Willow transparent flexible glass (Dow Corning). The anode250 is comprised of ITO. Light generated from the flexible light emitter240 is emitted through the transparent anode 250 and barrier layer 280such that the device has a “top” light emitting configuration.

Hydrogel or Adhesive Layer

In some embodiments, a transparent hydrogel, silicone membrane oradhesive layer such as Dow silicone PSA or double-sided adhesive tapelayer is applied between the light emitting surface of the lamp toattach it to the surface to be irradiated, such as a skin or tissuesurface. This layer may be a single use disposable in cases wherecertain microorganisms are able to penetrate the hydrogel, silicone oradhesive layer. As such, the hydrogel, silicone or adhesive layer is notsuitable for re-use due to the infection control requirements andnon-transmittal of infection back to the user or subsequent users of thedevice. Alternatively, this layer may be used multiple times in caseswhere there is no microorganism penetration. For example, studies haveshown that certain bacteria do not grow on the surface of a hydrogel orsilicone layer. The hydrogel, silicone or adhesive layers may alsocontain additional anti-bacterials and substances suitable to enhancetissue regeneration for use in wound healing. The double-sidedtransparent adhesive tape may be, for example, 3M 9964 Clear PolyesterDiagnostic Microfluidic Medical Tape (3M.com). Various hydrogels andsilicones are available based on the adhesion requirements for variousskin types and wound dressings.

VIII. Control of Light Source

The light source is driven and controlled by an electronic circuit thatincludes, for example, a power supply, drive circuit and control module.For flexible light sources, the electronic circuit may be provided in aseparate housing electrically connected to the light source or may bebuilt into the flexible material that mounts the light source.

The power supply may be any power supply capable of supplying sufficientpower to activate the light source. The power supply may comprise adisposable or rechargeable battery, solar cell, fuel cell, an adapter,or may be powered by the power grid. The battery may be a printedbattery, flexible lithium-ion primary or secondary cells, carbonnanotube, electrochemical inks, or other flexible organic or inorganicprimary or secondary cells using non-toxic or limited toxicitymaterials. In some embodiments, the battery is roll-to-roll printed onthe lamp substrate. The light source is preferably driven by DC orpulsed DC. One skilled in the art will understand that the outputvoltages and current levels of the DC or pulsed DC control the peakoutput of each layer of the device, which in combination with thetreatment time control the dose.

FIG. 31 is a block diagram of an exemplary system in which the lightsource and light source drive circuit are controlled by amicrocontroller (discussed below) in accordance with a preprogrammedtreatment cycle that provides a sequence of light at a fixed dose orwavelength based on the patch configuration. For example, themicrocontroller may be preprogrammed for treatment of a specificdisorder, such as acne. The microcontroller may control the light sourceby adjusting the activation and deactivation of the light source,voltage, current, light wavelength, pulse width, duty factor, and lighttreatment time. One skilled in the art will appreciate that otheroperating parameters may also be controlled by the microcontroller inaccordance with the present invention.

The microcontroller is also connected to one or more I/O devices, suchas an LED that provides an indication of whether the light source ison/off or an audio buzzer that alerts the user upon completion of aparticular treatment. An on/off switch may also be provided to power thelight source.

The system may be used in a number of treatment (irradiation) sessionsthat together result in an overall treatment time. For such cases, themicrocontroller may include at least one timer configured to measuresession time and overall treatment time or both. The timer may be usedsimply to monitor the session time or overall treatment time or may beused to deactivate the light source after completion of a session oroverall treatment. The timer may also be used to provide sequentialtreatments for automatic dosing in a wound care dressing, for example. Areal time clock associated with the system can monitor the treatmentsand track/manage treatment sequences and dosing.

FIG. 32 is a block diagram of an exemplary system in which the lightsource is controlled by an electronic circuit with one or more sensorsthat operate in a closed loop to provide feedback to a microcontrollerso as to dynamically control the light source during a treatment(irradiation) session. As can be seen, the electronic circuit is similarto that shown and described in connection with FIG. 31, with theaddition of one or more sensors that operate in a closed loop to providefeedback to the microcontroller. For example, in applications involvingthe photoeradication of P. acnes and other microorganisms, a photosensor could be used to detect porphyrins or other spectra related tothe inactivation of the bacteria that emit light within the red or otherwavelength portion of the fluorescent spectrum, as discussed above. Anexemplary sensor is a photo sensor used to detect porphyrins that emitlight within the red or other portion of the fluorescent spectrum inapplications involving the photoeradication of P. acnes with pulsedpurple or blue light or other spectra related to the inactivation of thebacteria. The photo sensor may comprise, for example, a photo detectorwith a sharp purple or blue notch filter and a bandpass filter that isused to detect the red or other wavelength portion of the fluorescentspectrum. This would be monitored during treatment and as it decayed toa preset threshold the pulsed purple or blue light stimulation would beturned off. From time to time, the system would turn on to see if theporphyrin levels would return to a high enough level to inducemicroorganism kill upon exposure to pulsed purple or blue light. Ofcourse, other types of sensors may also be used in accordance with theinvention. Certain sensors may be built into the layers of the device,while other sensors may be applied to the skin or other surface to beirradiated or may be built into the illuminator.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains. Also, as will be understood byone skilled in the art, for any and all purposes, all ranges disclosedherein also encompass any and all possible subranges and combinations ofsubranges thereof. Any listed range can be easily recognized assufficiently describing and enabling the same range being broken downinto at least equal halves, thirds, quarters, fifths, tenths, etc. Aswill also be understood by one skilled in the art, a range includes eachindividual member.

While the present invention has been described and illustratedhereinabove with reference to several exemplary embodiments, it shouldbe understood that various modifications could be made to theseembodiments without departing from the scope of the invention.Therefore, the present invention is not to be limited to the specificmethodologies or device configurations of the exemplary embodiments,except insofar as such limitations are included in the following claims.

What is claimed and desired to be secured by Letters Patent is as follows:
 1. A method for photoeradication of microorganisms from a target, the method comprising: obtaining test data for each of a plurality of experiments, wherein each experiment comprises irradiating test microorganisms with a plurality of light pulses having a wavelength that ranges from 380 nm to 500 nm, wherein the light pulses have a plurality of pulse parameters comprising a peak irradiance for each of the light pulses, a pulse duration for each of the light pulses, and an off time between each two adjacent light pulses, wherein the light pulses are provided at a radiant exposure that ranges from 0.5 J/cm² to 60 J/cm² during each of one or more irradiation sessions, and wherein the test data comprises a survival rate for the test microorganisms after irradiation with the light pulses; analyzing the test data to identify the pulse parameters for the light pulses and the radiant exposure for each of the irradiation sessions that result in a desired survival rate for the test microorganisms; and irradiating the microorganisms of the target with light pulses having the identified pulse parameters at the identified radiant exposure for each of the irradiation sessions so as to photoeradicate all or a portion of the microorganisms.
 2. The method of claim 1, wherein the microorganisms comprise one of a bacteria, a virus, or fungi.
 3. The method of claim 1, wherein the peak irradiance for each of the light pulses ranges from 0.3 mW/cm² to 60 mW/cm², the pulse duration for each of the light pulses ranges from 5 microseconds to 1,000 microseconds, and the off time between each two adjacent light pulses ranges from 10 microseconds to 1 second.
 4. The method of claim 3, wherein the light pulses are provided at a duty factor that ranges from 20% to 33% and a pulse repetition rate that ranges from 33 kHz to 40 kHz.
 5. The method of claim 1, wherein the microorganisms are associated with a photoactive molecule capable of photoeradication of all or a portion of the microorganisms.
 6. The method of claim 5, wherein the peak irradiance and the pulse duration for each of the light pulses is sufficient to optically excite the photoactive molecule, and wherein the off time between each two adjacent light pulses is sufficient to allow the photoactive molecule to return to a ground state creating an oxidation reaction that produces free radicals which subsequently destroy cellular structures of the microorganisms.
 7. The method of claim 5, wherein the microorganisms are suspended in a medium that is endogenous to a human body, wherein the medium contains the photoactive molecule.
 8. The method of claim 7, wherein the medium comprises human saliva.
 9. The method of claim 5, wherein the microorganisms are hosted within a plurality of cells that are endogenous to a human body, wherein the cells contain the photoactive molecule.
 10. The method of claim 9, wherein the cells comprise one of type II alveolar cells, respiratory epithelial cells, or human platelets.
 11. The method of claim 5, wherein the photoactive molecule comprises an exogenous photosensitizer administered to the target.
 12. The method of claim 11, wherein the photosensitizer is ingestible by a human body and comprises one of curcumin, aminolevulinic acid (ALA), or chloroquine and its derivatives.
 13. The method of claim 12, wherein the photosensitizer is topically applied to a human body and comprises one of curcumin, aminolevulinic acid (ALA), protoporphyrin IX (PPIX), coproporphyrin III (CP III), flavin mononucleotide (FMN), or nicotinamide adenine dinucleotide (NAD).
 14. The method of claim 5, wherein the irradiation sessions are timed to a depletion and recovery cycle of the photoactive molecule.
 15. The method of claim 1, wherein the irradiation sessions are timed to a replication cycle of the microorganisms.
 16. The method of claim 1, wherein the light pulses are applied to the target using a plurality of printed light emitting diodes incorporated into a respirator mask.
 17. The method of claim 1, wherein the light pulses are applied to the target using a plurality of printed light emitting diodes incorporated into a nasal applicator.
 18. The method of claim 1, wherein the light pulses are applied to the target using one or more light emitting diodes incorporated into a nasal applicator.
 19. The method of claim 1, wherein the light pulses are applied to the target using a plurality of printed light emitting diodes incorporated into a light device placed externally on a cheek region of a human body.
 20. The method of claim 1, wherein the light pulses are applied to the target using a plurality of printed light emitting diodes incorporated into a light device placed externally on an inner wrist region of a human body.
 21. The method of claim 1, wherein the light pulses are applied to the target using a plurality of printed light emitting diodes incorporated into a light device placed internally within an oral cavity of a human body.
 22. The method of claim 1, wherein the target comprises a plurality of cells infected by the microorganisms.
 23. The method of claim 1, wherein the target comprises an environment contaminated with the microorganisms. 